The Drosophila gene collection: Identification of putative full-length cDNAs for 70 percent of D. melanogaster genes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Stapleton, Mark; Liao, Guochun; Brokstein, Peter

2002-08-12

Collections of full-length nonredundant cDNA clones are critical reagents for functional genomics. The first step toward these resources is the generation and single-pass sequencing of cDNA libraries that contain a high proportion of full-length clones. The first release of the Drosophila Gene Collection Release 1 (DGCr1) was produced from six libraries representing various tissues, developmental stages, and the cultured S2 cell line. Nearly 80,000 random 5prime expressed sequence tags (EST) from these libraries were collapsed into a nonredundant set of 5849 cDNAs, corresponding to {approx}40 percent of the 13,474 predicted genes in Drosophila. To obtain cDNA clones representing the remainingmore » genes, we have generated an additional 157,835 5prime ESTs from two previously existing and three new libraries. One new library is derived from adult testis, a tissue we previously did not exploit for gene discovery; two new cap-trapped normalized libraries are derived from 0-22hr embryos and adult heads. Taking advantage of the annotated D. melanogaster genome sequence, we clustered the ESTs by aligning them to the genome. Clusters that overlap genes not already represented by cDNA clones in the DGCr1 were analyzed further, and putative full-length clones were selected for inclusion in the new DGC. This second release of the DGC (DGCr2) contains 5061 additional clones, extending the collection to 10,910 cDNAs representing >70 percent of the predicted genes in Drosophila.« less

Lack of hormone binding in COS-7 cells expressing a mutated growth hormone receptor found in Laron dwarfism.

PubMed Central

Edery, M; Rozakis-Adcock, M; Goujon, L; Finidori, J; Lévi-Meyrueis, C; Paly, J; Djiane, J; Postel-Vinay, M C; Kelly, P A

1993-01-01

A single point mutation in the growth hormone (GH) receptor gene generating a Phe-->Ser substitution in the extracellular binding domain of the receptor has been identified in one family with Laron type dwarfism. The mutation was introduced by site-directed mutagenesis into cDNAs encoding the full-length rabbit GH receptor and the extracellular domain or binding protein (BP) of the human and rabbit GH receptor, and also in cDNAs encoding the full length and the extracellular domain of the related rabbit prolactin (PRL) receptor. All constructs were transiently expressed in COS-7 cells. Both wild type and mutant full-length rabbit GH and PRL receptors, as well as GH and prolactin BPs (wild type and mutant), were detected by Western blot in cell membranes and concentrated culture media, respectively. Immunofluorescence studies showed that wild type and mutant full-length GH receptors had the same cell surface and intracellular distribution and were expressed with comparable intensities. In contrast, all mutant forms (full-length receptors or BPs), completely lost their modify the synthesis ligand. These results clearly demonstrate that this point mutation (patients with Laron syndrome) does not modify the synthesis or the intracellular pathway of receptor proteins, but rather abolishes ability of the receptor or BP to bind GH and is thus responsible for the extreme GH resistance in these patients. Images PMID:8450064

Particle infectivity of HIV-1 full-length genome infectious molecular clones in a subtype C heterosexual transmission pair following high fidelity amplification and unbiased cloning

DOE Office of Scientific and Technical Information (OSTI.GOV)

Deymier, Martin J., E-mail: mdeymie@emory.edu; Claiborne, Daniel T., E-mail: dclaibo@emory.edu; Ende, Zachary, E-mail: zende@emory.edu

The high genetic diversity of HIV-1 impedes high throughput, large-scale sequencing and full-length genome cloning by common restriction enzyme based methods. Applying novel methods that employ a high-fidelity polymerase for amplification and an unbiased fusion-based cloning strategy, we have generated several HIV-1 full-length genome infectious molecular clones from an epidemiologically linked transmission pair. These clones represent the transmitted/founder virus and phylogenetically diverse non-transmitted variants from the chronically infected individual's diverse quasispecies near the time of transmission. We demonstrate that, using this approach, PCR-induced mutations in full-length clones derived from their cognate single genome amplicons are rare. Furthermore, all eight non-transmittedmore » genomes tested produced functional virus with a range of infectivities, belying the previous assumption that a majority of circulating viruses in chronic HIV-1 infection are defective. Thus, these methods provide important tools to update protocols in molecular biology that can be universally applied to the study of human viral pathogens. - Highlights: • Our novel methodology demonstrates accurate amplification and cloning of full-length HIV-1 genomes. • A majority of plasma derived HIV variants from a chronically infected individual are infectious. • The transmitted/founder was more infectious than the majority of the variants from the chronically infected donor.« less

Purification of full-length VP22 from cells infected with HSV-1: A two-pronged approach for the solubilization and purification of viral proteins for use in biochemical studies

PubMed Central

Dewberry, Ebony J.; Dunkerley, Eric; Duffy, Carol

2012-01-01

Summary VP22, encoded by the UL49 gene, is one of the most abundant proteins of the herpes simplex virus type 1 (HSV-1) tegument and has been shown to be important for virus replication and spread. However, the exact role(s) played by VP22 in the HSV-1 replication cycle have yet to be delineated. The lack of a procedure to purify full-length VP22 has limited molecular studies on VP22 function. A procedure was developed for the purification of soluble, full-length VP22 from cells infected with HSV-1. A recombinant virus encoding His-tagged VP22 was generated and found to express VP22 at levels comparable to the wild type virus upon infection of Vero cells. By experimenting with a wide variety of cell lysis buffer conditions, several buffers that promote the solubility of full-length VP22 were identified. Buffers that gave the highest levels of solubility were then used in immobilized metal ion affinity chromatography experiments to identify conditions that provided the greatest level of VP22 binding and recovery from cobalt and nickel affinity resins. Using this strategy soluble, full-length VP22 was purified from cells infected with HSV-1. PMID:22569534

Stroma-induced Jagged1 expression drives PC3 prostate cancer cell migration; disparate effects of RIP-generated proteolytic fragments on cell behaviour and Notch signaling

DOE Office of Scientific and Technical Information (OSTI.GOV)

Delury, Craig, E-mail: c.delury@lancaster.ac.uk; Hart, Claire, E-mail: claire.hart@manchester.ac.uk; Brown, Mick, E-mail: michael.brown@ics.manchester.ac.uk

The Notch ligand Jagged1 is subject to regulated intramembrane proteolysis (RIP) which yields a soluble ectodomain (sJag) and a soluble Jagged1 intracellular domain (JICD). The full-length Jagged1 protein enhances prostate cancer (PCa) cell proliferation and is highly expressed in metastatic cells. However, little is known regarding the mechanisms by which Jagged1 or its RIP-generated fragments might promote PCa bone metastasis. In the current study we show that bone marrow stroma (BMS) induces Jagged1 expression in bone metastatic prostate cancer PC3 cells and that this enhanced expression is mechanistically linked to the promotion of cell migration. We also show that RIP-generatedmore » Jagged1 fragments exert disparate effects on PC3 cell behaviour and Notch signaling. In conclusion, the expression of both the full-length ligand and its RIP-generated fragments must be considered in tandem when attempting to regulate Jagged1 as a possible PCa therapy. - Highlights: • Bone marrow stroma induces Jagged1 expression in prostate cancer (PCa) PC3 cells. • This enhanced expression of full-length Jagged1 is required for PC3 cell migration. • Proteolytic fragments of Jagged1 exert disparate effects on PC3 cell behaviour. • Effects of fragments on cell behaviour do not correlate with Notch signaling. • Effects of Jagged1 and its fragments on PCa metastasis likely to be complex.« less

Near Full-Length Identification of a Novel HIV-1 CRF01_AE/B/C Recombinant in Northern Myanmar.

PubMed

Zhou, Yan-Heng; Chen, Xin; Liang, Yue-Bo; Pang, Wei; Qin, Wei-Hong; Zhang, Chiyu; Zheng, Yong-Tang

2015-08-01

The Myanmar-China border appears to be the "hot spot" region for the occurrence of HIV-1 recombination. The majority of the previous analyses of HIV-1 recombination were based on partial genomic sequences, which obviously cannot reflect the reality of the genetic diversity of HIV-1 in this area well. Here, we present a near full-length characterization of a novel HIV-1 CRF01_AE/B/C recombinant isolated from a long-distance truck driver in Northern Myanmar. It is the first description of a near full-length genomic sequence in Myanmar since 2003, and might be one of the most complicated HIV-1 chimeras ever detected in Myanmar, containing four CRF01_AE, six B segments, and five C segments separated by 14 breakpoints throughout its genome. The discovery and characterization of this new CRF01_AE/B/C recombinant indicate that intersubtype recombination is ongoing in Myanmar, continuously generating new forms of HIV-1. More work based on near full-length sequence analyses is urgently needed to better understand the genetic diversity of HIV-1 in these regions.

Isoform Sequencing Provides a More Comprehensive View of the Panax ginseng Transcriptome.

PubMed

Jo, Ick-Hyun; Lee, Jinsu; Hong, Chi Eun; Lee, Dong Jin; Bae, Wonsil; Park, Sin-Gi; Ahn, Yong Ju; Kim, Young Chang; Kim, Jang Uk; Lee, Jung Woo; Hyun, Dong Yun; Rhee, Sung-Keun; Hong, Chang Pyo; Bang, Kyong Hwan; Ryu, Hojin

2017-09-15

Korean ginseng ( Panax ginseng C.A. Meyer) has been widely used for medicinal purposes and contains potent plant secondary metabolites, including ginsenosides. To obtain transcriptomic data that offers a more comprehensive view of functional genomics in P. ginseng , we generated genome-wide transcriptome data from four different P. ginseng tissues using PacBio isoform sequencing (Iso-Seq) technology. A total of 135,317 assembled transcripts were generated with an average length of 3.2 kb and high assembly completeness. Of those unigenes, 67.5% were predicted to be complete full-length (FL) open reading frames (ORFs) and exhibited a high gene annotation rate. Furthermore, we successfully identified unique full-length genes involved in triterpenoid saponin synthesis and plant hormonal signaling pathways, including auxin and cytokinin. Studies on the functional genomics of P. ginseng seedlings have confirmed the rapid upregulation of negative feed-back loops by auxin and cytokinin signaling cues. The conserved evolutionary mechanisms in the auxin and cytokinin canonical signaling pathways of P. ginseng are more complex than those in Arabidopsis thaliana . Our analysis also revealed a more detailed view of transcriptome-wide alternative isoforms for 88 genes. Finally, transposable elements (TEs) were also identified, suggesting transcriptional activity of TEs in P. ginseng . In conclusion, our results suggest that long-read, full-length or partial-unigene data with high-quality assemblies are invaluable resources as transcriptomic references in P. ginseng and can be used for comparative analyses in closely related medicinal plants.

Recovery of infectious virus from full-length cowpox virus (CPXV) DNA cloned as a bacterial artificial chromosome (BAC)

PubMed Central

2011-01-01

Transmission from pet rats and cats to humans as well as severe infection in felids and other animal species have recently drawn increasing attention to cowpox virus (CPXV). We report the cloning of the entire genome of cowpox virus strain Brighton Red (BR) as a bacterial artificial chromosome (BAC) in Escherichia coli and the recovery of infectious virus from cloned DNA. Generation of a full-length CPXV DNA clone was achieved by first introducing a mini-F vector, which allows maintenance of large circular DNA in E. coli, into the thymidine kinase locus of CPXV by homologous recombination. Circular replication intermediates were then electroporated into E. coli DH10B cells. Upon successful establishment of the infectious BR clone, we modified the full-length clone such that recombination-mediated excision of bacterial sequences can occur upon transfection in eukaryotic cells. This self-excision of the bacterial replicon is made possible by a sequence duplication within mini-F sequences and allows recovery of recombinant virus progeny without remaining marker or vector sequences. The in vitro growth properties of viruses derived from both BAC clones were determined and found to be virtually indistinguishable from those of parental, wild-type BR. Finally, the complete genomic sequence of the infectious clone was determined and the cloned viral genome was shown to be identical to that of the parental virus. In summary, the generated infectious clone will greatly facilitate studies on individual genes and pathogenesis of CPXV. Moreover, the vector potential of CPXV can now be more systematically explored using this newly generated tool. PMID:21314965

In vitro characterization of felid herpesvirus 1 (FHV-1) mutants generated by recombineering in a recombinant BAC vector

USDA-ARS?s Scientific Manuscript database

Felid herpesvirus 1 (FHV-1) mutants were constructed using two-step Red-mediated recombination techniques based on a virulent full-length FHV-1 BAC clone. The individual mutant viruses generated were deficient in glycoprotein C (gC), glycoprotein E (gE),US3 serine/threonine protein kinase (PK), or b...

A survey of the sorghum transcriptome using single-molecule long reads

DOE PAGES

Abdel-Ghany, Salah E.; Hamilton, Michael; Jacobi, Jennifer L.; ...

2016-06-24

Alternative splicing and alternative polyadenylation (APA) of pre-mRNAs greatly contribute to transcriptome diversity, coding capacity of a genome and gene regulatory mechanisms in eukaryotes. Second-generation sequencing technologies have been extensively used to analyse transcriptomes. However, a major limitation of short-read data is that it is difficult to accurately predict full-length splice isoforms. Here we sequenced the sorghum transcriptome using Pacific Biosciences single-molecule real-time long-read isoform sequencing and developed a pipeline called TAPIS (Transcriptome Analysis Pipeline for Isoform Sequencing) to identify full-length splice isoforms and APA sites. Our analysis reveals transcriptome-wide full-length isoforms at an unprecedented scale with over 11,000 novelmore » splice isoforms. Additionally, we uncover APA ofB11,000 expressed genes and more than 2,100 novel genes. Lastly, these results greatly enhance sorghum gene annotations and aid in studying gene regulation in this important bioenergy crop. The TAPIS pipeline will serve as a useful tool to analyse Iso-Seq data from any organism.« less

A survey of the sorghum transcriptome using single-molecule long reads

PubMed Central

Abdel-Ghany, Salah E.; Hamilton, Michael; Jacobi, Jennifer L.; Ngam, Peter; Devitt, Nicholas; Schilkey, Faye; Ben-Hur, Asa; Reddy, Anireddy S. N.

2016-01-01

Alternative splicing and alternative polyadenylation (APA) of pre-mRNAs greatly contribute to transcriptome diversity, coding capacity of a genome and gene regulatory mechanisms in eukaryotes. Second-generation sequencing technologies have been extensively used to analyse transcriptomes. However, a major limitation of short-read data is that it is difficult to accurately predict full-length splice isoforms. Here we sequenced the sorghum transcriptome using Pacific Biosciences single-molecule real-time long-read isoform sequencing and developed a pipeline called TAPIS (Transcriptome Analysis Pipeline for Isoform Sequencing) to identify full-length splice isoforms and APA sites. Our analysis reveals transcriptome-wide full-length isoforms at an unprecedented scale with over 11,000 novel splice isoforms. Additionally, we uncover APA of ∼11,000 expressed genes and more than 2,100 novel genes. These results greatly enhance sorghum gene annotations and aid in studying gene regulation in this important bioenergy crop. The TAPIS pipeline will serve as a useful tool to analyse Iso-Seq data from any organism. PMID:27339290

Performance of an Axisymmetric Rocket Based Combined Cycle Engine During Rocket Only Operation Using Linear Regression Analysis

NASA Technical Reports Server (NTRS)

Smith, Timothy D.; Steffen, Christopher J., Jr.; Yungster, Shaye; Keller, Dennis J.

1998-01-01

The all rocket mode of operation is shown to be a critical factor in the overall performance of a rocket based combined cycle (RBCC) vehicle. An axisymmetric RBCC engine was used to determine specific impulse efficiency values based upon both full flow and gas generator configurations. Design of experiments methodology was used to construct a test matrix and multiple linear regression analysis was used to build parametric models. The main parameters investigated in this study were: rocket chamber pressure, rocket exit area ratio, injected secondary flow, mixer-ejector inlet area, mixer-ejector area ratio, and mixer-ejector length-to-inlet diameter ratio. A perfect gas computational fluid dynamics analysis, using both the Spalart-Allmaras and k-omega turbulence models, was performed with the NPARC code to obtain values of vacuum specific impulse. Results from the multiple linear regression analysis showed that for both the full flow and gas generator configurations increasing mixer-ejector area ratio and rocket area ratio increase performance, while increasing mixer-ejector inlet area ratio and mixer-ejector length-to-diameter ratio decrease performance. Increasing injected secondary flow increased performance for the gas generator analysis, but was not statistically significant for the full flow analysis. Chamber pressure was found to be not statistically significant.

Evidence of structurally continuous collagen fibrils in tendons.

PubMed

Svensson, Rene B; Herchenhan, Andreas; Starborg, Tobias; Larsen, Michael; Kadler, Karl E; Qvortrup, Klaus; Magnusson, S Peter

2017-03-01

Tendons transmit muscle-generated force through an extracellular matrix of aligned collagen fibrils. The force applied by the muscle at one end of a microscopic fibril has to be transmitted through the macroscopic length of the tendon by mechanisms that are poorly understood. A key element in this structure-function relationship is the collagen fibril length. During embryogenesis short fibrils are produced but they grow rapidly with maturation. There is some controversy regarding fibril length in adult tendon, with mechanical data generally supporting discontinuity while structural investigations favor continuity. This study initially set out to trace the full length of individual fibrils in adult human tendons, using serial block face-scanning electron microscopy. But even with this advanced technique the required length could not be covered. Instead a statistical approach was used on a large volume of fibrils in shorter image stacks. Only a single end was observed after tracking 67.5mm of combined fibril lengths, in support of fibril continuity. To shed more light on this observation, the full length of a short tendon (mouse stapedius, 125μm) was investigated and continuity of individual fibrils was confirmed. In light of these results, possible mechanisms that could reconcile the opposing findings on fibril continuity are discussed. Connective tissues hold all parts of the body together and are mostly constructed from thin threads of the protein collagen (called fibrils). Connective tissues provide mechanical strength and one of the most demanding tissues in this regard are tendons, which transmit the forces generated by muscles. The length of the collagen fibrils is essential to the mechanical strength and to the type of damage the tissue may experience (slippage of short fibrils or breakage of longer ones). This in turn is important for understanding the repair processes after such damage occurs. Currently the issue of fibril length is contentious, but this study provides evidence that the fibrils are extremely long and likely continuous. Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

The contribution of 700,000 ORF sequence tags to the definition of the human transcriptome

PubMed Central

Camargo, Anamaria A.; Samaia, Helena P. B.; Dias-Neto, Emmanuel; Simão, Daniel F.; Migotto, Italo A.; Briones, Marcelo R. S.; Costa, Fernando F.; Aparecida Nagai, Maria; Verjovski-Almeida, Sergio; Zago, Marco A.; Andrade, Luis Eduardo C.; Carrer, Helaine; El-Dorry, Hamza F. A.; Espreafico, Enilza M.; Habr-Gama, Angelita; Giannella-Neto, Daniel; Goldman, Gustavo H.; Gruber, Arthur; Hackel, Christine; Kimura, Edna T.; Maciel, Rui M. B.; Marie, Suely K. N.; Martins, Elizabeth A. L.; Nóbrega, Marina P.; Paçó-Larson, Maria Luisa; Pardini, Maria Inês M. C.; Pereira, Gonçalo G.; Pesquero, João Bosco; Rodrigues, Vanderlei; Rogatto, Silvia R.; da Silva, Ismael D. C. G.; Sogayar, Mari C.; Sonati, Maria de Fátima; Tajara, Eloiza H.; Valentini, Sandro R.; Alberto, Fernando L.; Amaral, Maria Elisabete J.; Aneas, Ivy; Arnaldi, Liliane A. T.; de Assis, Angela M.; Bengtson, Mário Henrique; Bergamo, Nadia Aparecida; Bombonato, Vanessa; de Camargo, Maria E. R.; Canevari, Renata A.; Carraro, Dirce M.; Cerutti, Janete M.; Corrêa, Maria Lucia C.; Corrêa, Rosana F. R.; Costa, Maria Cristina R.; Curcio, Cyntia; Hokama, Paula O. M.; Ferreira, Ari J. S.; Furuzawa, Gilberto K.; Gushiken, Tsieko; Ho, Paulo L.; Kimura, Elza; Krieger, José E.; Leite, Luciana C. C.; Majumder, Paromita; Marins, Mozart; Marques, Everaldo R.; Melo, Analy S. A.; Melo, Monica; Mestriner, Carlos Alberto; Miracca, Elisabete C.; Miranda, Daniela C.; Nascimento, Ana Lucia T. O.; Nóbrega, Francisco G.; Ojopi, Élida P. B.; Pandolfi, José Rodrigo C.; Pessoa, Luciana G.; Prevedel, Aline C.; Rahal, Paula; Rainho, Claudia A.; Reis, Eduardo M. R.; Ribeiro, Marcelo L.; da Rós, Nancy; de Sá, Renata G.; Sales, Magaly M.; Sant'anna, Simone Cristina; dos Santos, Mariana L.; da Silva, Aline M.; da Silva, Neusa P.; Silva, Wilson A.; da Silveira, Rosana A.; Sousa, Josane F.; Stecconi, Daniella; Tsukumo, Fernando; Valente, Valéria; Soares, Fernando; Moreira, Eloisa S.; Nunes, Diana N.; Correa, Ricardo G.; Zalcberg, Heloisa; Carvalho, Alex F.; Reis, Luis F. L.; Brentani, Ricardo R.; Simpson, Andrew J. G.; de Souza, Sandro J.

2001-01-01

Open reading frame expressed sequences tags (ORESTES) differ from conventional ESTs by providing sequence data from the central protein coding portion of transcripts. We generated a total of 696,745 ORESTES sequences from 24 human tissues and used a subset of the data that correspond to a set of 15,095 full-length mRNAs as a means of assessing the efficiency of the strategy and its potential contribution to the definition of the human transcriptome. We estimate that ORESTES sampled over 80% of all highly and moderately expressed, and between 40% and 50% of rarely expressed, human genes. In our most thoroughly sequenced tissue, the breast, the 130,000 ORESTES generated are derived from transcripts from an estimated 70% of all genes expressed in that tissue, with an equally efficient representation of both highly and poorly expressed genes. In this respect, we find that the capacity of the ORESTES strategy both for gene discovery and shotgun transcript sequence generation significantly exceeds that of conventional ESTs. The distribution of ORESTES is such that many human transcripts are now represented by a scaffold of partial sequences distributed along the length of each gene product. The experimental joining of the scaffold components, by reverse transcription–PCR, represents a direct route to transcript finishing that may represent a useful alternative to full-length cDNA cloning. PMID:11593022

The contribution of 700,000 ORF sequence tags to the definition of the human transcriptome.

PubMed

Camargo, A A; Samaia, H P; Dias-Neto, E; Simão, D F; Migotto, I A; Briones, M R; Costa, F F; Nagai, M A; Verjovski-Almeida, S; Zago, M A; Andrade, L E; Carrer, H; El-Dorry, H F; Espreafico, E M; Habr-Gama, A; Giannella-Neto, D; Goldman, G H; Gruber, A; Hackel, C; Kimura, E T; Maciel, R M; Marie, S K; Martins, E A; Nobrega, M P; Paco-Larson, M L; Pardini, M I; Pereira, G G; Pesquero, J B; Rodrigues, V; Rogatto, S R; da Silva, I D; Sogayar, M C; Sonati, M F; Tajara, E H; Valentini, S R; Alberto, F L; Amaral, M E; Aneas, I; Arnaldi, L A; de Assis, A M; Bengtson, M H; Bergamo, N A; Bombonato, V; de Camargo, M E; Canevari, R A; Carraro, D M; Cerutti, J M; Correa, M L; Correa, R F; Costa, M C; Curcio, C; Hokama, P O; Ferreira, A J; Furuzawa, G K; Gushiken, T; Ho, P L; Kimura, E; Krieger, J E; Leite, L C; Majumder, P; Marins, M; Marques, E R; Melo, A S; Melo, M B; Mestriner, C A; Miracca, E C; Miranda, D C; Nascimento, A L; Nobrega, F G; Ojopi, E P; Pandolfi, J R; Pessoa, L G; Prevedel, A C; Rahal, P; Rainho, C A; Reis, E M; Ribeiro, M L; da Ros, N; de Sa, R G; Sales, M M; Sant'anna, S C; dos Santos, M L; da Silva, A M; da Silva, N P; Silva, W A; da Silveira, R A; Sousa, J F; Stecconi, D; Tsukumo, F; Valente, V; Soares, F; Moreira, E S; Nunes, D N; Correa, R G; Zalcberg, H; Carvalho, A F; Reis, L F; Brentani, R R; Simpson, A J; de Souza, S J; Melo, M

2001-10-09

Open reading frame expressed sequences tags (ORESTES) differ from conventional ESTs by providing sequence data from the central protein coding portion of transcripts. We generated a total of 696,745 ORESTES sequences from 24 human tissues and used a subset of the data that correspond to a set of 15,095 full-length mRNAs as a means of assessing the efficiency of the strategy and its potential contribution to the definition of the human transcriptome. We estimate that ORESTES sampled over 80% of all highly and moderately expressed, and between 40% and 50% of rarely expressed, human genes. In our most thoroughly sequenced tissue, the breast, the 130,000 ORESTES generated are derived from transcripts from an estimated 70% of all genes expressed in that tissue, with an equally efficient representation of both highly and poorly expressed genes. In this respect, we find that the capacity of the ORESTES strategy both for gene discovery and shotgun transcript sequence generation significantly exceeds that of conventional ESTs. The distribution of ORESTES is such that many human transcripts are now represented by a scaffold of partial sequences distributed along the length of each gene product. The experimental joining of the scaffold components, by reverse transcription-PCR, represents a direct route to transcript finishing that may represent a useful alternative to full-length cDNA cloning.

Protein expression, characterization and activity comparisons of wild type and mutant DUSP5 proteins

DOE PAGES

Nayak, Jaladhi; Gastonguay, Adam J.; Talipov, Marat R.; ...

2014-12-18

Background: The mitogen-activated protein kinases (MAPKs) pathway is critical for cellular signaling, and proteins such as phosphatases that regulate this pathway are important for normal tissue development. Based on our previous work on dual specificity phosphatase-5 (DUSP5), and its role in embryonic vascular development and disease, we hypothesized that mutations in DUSP5 will affect its function. Results: In this study, we tested this hypothesis by generating full-length glutathione-S-transferase-tagged DUSP5 and serine 147 proline mutant (S147P) proteins from bacteria. Light scattering analysis, circular dichroism, enzymatic assays and molecular modeling approaches have been performed to extensively characterize the protein form and function.more » We demonstrate that both proteins are active and, interestingly, the S147P protein is hypoactive as compared to the DUSP5 WT protein in two distinct biochemical substrate assays. Furthermore, due to the novel positioning of the S147P mutation, we utilize computational modeling to reconstruct full-length DUSP5 and S147P to predict a possible mechanism for the reduced activity of S147P. Conclusion: Taken together, this is the first evidence of the generation and characterization of an active, full-length, mutant DUSP5 protein which will facilitate future structure-function and drug development-based studies.« less

Characterization and long term operation of a novel superconducting undulator with 15 mm period length in a synchrotron light source

NASA Astrophysics Data System (ADS)

Casalbuoni, S.; Cecilia, A.; Gerstl, S.; Glamann, N.; Grau, A. W.; Holubek, T.; Meuter, C.; de Jauregui, D. Saez; Voutta, R.; Boffo, C.; Gerhard, Th.; Turenne, M.; Walter, W.

2016-11-01

A new cryogen-free full scale (1.5 m long) superconducting undulator with a period length of 15 mm (SCU15) has been successfully tested in the ANKA storage ring. This represents a very important milestone in the development of superconducting undulators for third and fourth generation light sources carried on by the collaboration between the Karlsruhe Institute of Technology and the industrial partner Babcock Noell GmbH. SCU15 is the first full length device worldwide that with beam reaches a higher peak field than what expected with the same geometry (vacuum gap and period length) with an ideal cryogenic permanent magnet undulator built with the best material available PrFeB. After a summary on the design and main parameters of the device, we present here the characterization in terms of spectral properties and the long term operation of the SCU15 in the ANKA storage ring.

EXPRESSION AND CHARACTERIZATION OF FULL-LENGTH HUMAN HEME OXYGENASE-1: PRESENCE OF INTACT MEMBRANE-BINDING REGION LEADS TO INCREASED BINDING AFFINITY FOR NADPH-CYTOCHROME P450 REDUCTASE

PubMed Central

Huber, Warren J.; Backes, Wayne L.

2009-01-01

Heme oxygenase (HO) is the chief regulatory enzyme in the oxidative degradation of heme to biliverdin. In the process of heme degradation, this NADPH and cytochrome P450 reductase (CPR)-dependent oxidation of heme also releases free iron and carbon monoxide. Much of the recent research involving heme oxygenase is done using a 30-kDa soluble form of the enzyme, which lacks the membrane binding region (C-terminal 23 amino acids). The goal of this study was to express and purify a full-length human HO-1 (hHO-1) protein; however, due to the lability of the full-length form, a rapid purification procedure was required. This was accomplished by use of a GST-tagged hHO-1 construct. Although the procedure permitted the generation of a full-length HO-1, this form was contaminated with a 30-kDa degradation product that could not be eliminated. Therefore, we attempted to remove a putative secondary thrombin cleavage site by a conservative mutation of amino acid 254, which replaces lysine with arginine. This mutation allowed the expression and purification of a full length hHO-1 protein. Unlike wild-type HO-1, the K254R mutant could be purified to a single 32-kDa protein capable of degrading heme at the same rate as the wild-type enzyme. The K254R full-length form had a specific activity of ~200–225 nmol bilirubin hr−1nmol−1 HO-1 as compared to ~140–150 nmol bilirubin hr−1nmol−1 for the WT form, which contains the 30-kDa contaminant. This is a 2–3-fold increase from the previously reported soluble 30-kDa HO-1, suggesting that the C-terminal 23 amino acids are essential for maximal catalytic activity. Because the membrane spanning domain is present, the full-length hHO-1 has the potential to incorporate into phospholipid membranes, which can be reconstituted at known concentrations, in combination with other ER-resident enzymes. PMID:17915953

Expression and characterization of full-length human heme oxygenase-1: the presence of intact membrane-binding region leads to increased binding affinity for NADPH cytochrome P450 reductase.

PubMed

Huber, Warren J; Backes, Wayne L

2007-10-30

Heme oxygenase-1 (HO-1) is the chief regulatory enzyme in the oxidative degradation of heme to biliverdin. In the process of heme degradation, HO-1 receives the electrons necessary for catalysis from the flavoprotein NADPH cytochrome P450 reductase (CPR), releasing free iron and carbon monoxide. Much of the recent research involving heme oxygenase has been done using a 30 kDa soluble form of the enzyme, which lacks the membrane binding region (C-terminal 23 amino acids). The goal of this study was to express and purify a full-length human HO-1 (hHO-1) protein; however, due to the lability of the full-length form, a rapid purification procedure was required. This was accomplished by use of a glutathione-s-transferase (GST)-tagged hHO-1 construct. Although the procedure permitted the generation of a full-length HO-1, this form was contaminated with a 30 kDa degradation product that could not be eliminated. Therefore, attempts were made to remove a putative secondary thrombin cleavage site by a conservative mutation of amino acid 254, which replaces arginine with lysine. This mutation allowed the expression and purification of a full-length hHO-1 protein. Unlike wild type (WT) HO-1, the R254K mutant could be purified to a single 32 kDa protein capable of degrading heme at the same rate as the WT enzyme. The R254K full-length form had a specific activity of approximately 200-225 nmol of bilirubin h-1 nmol-1 HO-1 as compared to approximately 140-150 nmol of bilirubin h-1 nmol-1 for the WT form, which contains the 30 kDa contaminant. This is a 2-3-fold increase from the previously reported soluble 30 kDa HO-1, suggesting that the C-terminal 23 amino acids are essential for maximal catalytic activity. Because the membrane-spanning domain is present, the full-length hHO-1 has the potential to incorporate into phospholipid membranes, which can be reconstituted at known concentrations, in combination with other endoplasmic reticulum resident enzymes.

Rapid Sequencing of Complete env Genes from Primary HIV-1 Samples.

PubMed

Laird Smith, Melissa; Murrell, Ben; Eren, Kemal; Ignacio, Caroline; Landais, Elise; Weaver, Steven; Phung, Pham; Ludka, Colleen; Hepler, Lance; Caballero, Gemma; Pollner, Tristan; Guo, Yan; Richman, Douglas; Poignard, Pascal; Paxinos, Ellen E; Kosakovsky Pond, Sergei L; Smith, Davey M

2016-07-01

The ability to study rapidly evolving viral populations has been constrained by the read length of next-generation sequencing approaches and the sampling depth of single-genome amplification methods. Here, we develop and characterize a method using Pacific Biosciences' Single Molecule, Real-Time (SMRT®) sequencing technology to sequence multiple, intact full-length human immunodeficiency virus-1 env genes amplified from viral RNA populations circulating in blood, and provide computational tools for analyzing and visualizing these data.

Characterization of a full-length infectious cDNA clone and a GFP reporter derivative of the oncolytic picornavirus SVV-001.

PubMed

Poirier, John T; Reddy, P Seshidhar; Idamakanti, Neeraja; Li, Shawn S; Stump, Kristine L; Burroughs, Kevin D; Hallenbeck, Paul L; Rudin, Charles M

2012-12-01

Seneca Valley virus (SVV-001) is an oncolytic picornavirus with selective tropism for a subset of human cancers with neuroendocrine differentiation. To characterize further the specificity of SVV-001 and its patterns and kinetics of intratumoral spread, bacterial plasmids encoding a cDNA clone of the full-length wild-type virus and a derivative virus expressing GFP were generated. The full-length cDNA of the SVV-001 RNA genome was cloned into a bacterial plasmid under the control of the T7 core promoter sequence to create an infectious cDNA clone, pNTX-09. A GFP reporter virus cDNA clone, pNTX-11, was then generated by cloning a fusion protein of GFP and the 2A protein from foot-and-mouth disease virus immediately following the native SVV-001 2A sequence. Recombinant GFP-expressing reporter virus, SVV-GFP, was rescued from cells transfected with in vitro RNA transcripts from pNTX-11 and propagated in cell culture. The proliferation kinetics of SVV-001 and SVV-GFP were indistinguishable. The SVV-GFP reporter virus was used to determine that a subpopulation of permissive cells is present in small-cell lung cancer cell lines previously thought to lack permissivity to SVV-001. Finally, it was shown that SVV-GFP administered to tumour-bearing animals homes in to and infects tumours whilst having no detectable tropism for normal mouse tissues at 1×10(11) viral particles kg(-1), a dose equivalent to that administered in ongoing clinical trials. These infectious clones will be of substantial value in further characterizing the biology of this virus and as a backbone for the generation of additional oncolytic derivatives.

Gapped Spectral Dictionaries and Their Applications for Database Searches of Tandem Mass Spectra*

PubMed Central

Jeong, Kyowon; Kim, Sangtae; Bandeira, Nuno; Pevzner, Pavel A.

2011-01-01

Generating all plausible de novo interpretations of a peptide tandem mass (MS/MS) spectrum (Spectral Dictionary) and quickly matching them against the database represent a recently emerged alternative approach to peptide identification. However, the sizes of the Spectral Dictionaries quickly grow with the peptide length making their generation impractical for long peptides. We introduce Gapped Spectral Dictionaries (all plausible de novo interpretations with gaps) that can be easily generated for any peptide length thus addressing the limitation of the Spectral Dictionary approach. We show that Gapped Spectral Dictionaries are small thus opening a possibility of using them to speed-up MS/MS searches. Our MS-GappedDictionary algorithm (based on Gapped Spectral Dictionaries) enables proteogenomics applications (such as searches in the six-frame translation of the human genome) that are prohibitively time consuming with existing approaches. MS-GappedDictionary generates gapped peptides that occupy a niche between accurate but short peptide sequence tags and long but inaccurate full length peptide reconstructions. We show that, contrary to conventional wisdom, some high-quality spectra do not have good peptide sequence tags and introduce gapped tags that have advantages over the conventional peptide sequence tags in MS/MS database searches. PMID:21444829

In vitro replication of poliovirus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lubinski, J.M.

1986-01-01

Poliovirus is a member of the Picornaviridae whose genome is a single stranded RNA molecule of positive polarity surrounded by a proteinaceous capsid. Replication of poliovirus occurs via negative strand intermediates in infected cells using a virally encoded RNA-dependent RNA polymerase and host cell proteins. The authors have exploited the fact that complete cDNA copies of the viral genome when transfected onto susceptible cells generate virus. Utilizing the bacteriophage SP6 DNA dependent RNA polymerase system to synthesize negative strands in vitro and using these in an in vitro reaction the authors have generated full length infectious plus strands. Mutagenesis ofmore » the 5' and 3' ends of the negative and positive strands demonstrated that replication could occur either de novo or be extensions of the templates from their 3' ends or from nicks occurring during replication. The appearance of dimeric RNA molecules generated in these reactions was not dependent upon the same protein required for de novo initiation. Full length dimeric RNA molecules using a 5' /sup 32/P end-labelled oligo uridylic acid primer and positive strand template were demonstrated in vitro containing only the 35,000 Mr host protein and the viral RNA-dependent RNA polymerase. A model for generating positive strands without protein priming by cleavage of dimeric RNA molecules was proposed.« less

Recombinant production and film properties of full-length hornet silk proteins.

PubMed

Kambe, Yusuke; Sutherland, Tara D; Kameda, Tsunenori

2014-08-01

Full-length versions of the four main components of silk cocoons of Vespa simillima hornets, Vssilk1-4, were produced as recombinant proteins in Escherichia coli. In shake flasks, the recombinant Vssilk proteins yielded 160-330mg recombinant proteinl(-1). Films generated from solutions of single Vssilk proteins had a secondary structure similar to that of films generated from native hornet silk. The films made from individual recombinant hornet silk proteins had similar or enhanced mechanical performance compared with films generated from native hornet silk, possibly reflecting the homogeneity of the recombinant proteins. The pH-dependent changes in zeta (ζ) potential of each Vssilk film were measured, and isoelectric points (pI) of Vssilk1-4 were determined as 8.9, 9.1, 5.0 and 4.2, respectively. The pI of native hornet silk, a combination of the four Vssilk proteins, was 4.7, a value similar to that of Bombyx mori silkworm silk. Films generated from Vssilk1 and 2 had net positive charge under physiological conditions and showed significantly higher cell adhesion activity. It is proposed that recombinant hornet silk is a valuable new material with potential for cell culture applications. Copyright © 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Rapid Sequencing of Complete env Genes from Primary HIV-1 Samples

PubMed Central

Eren, Kemal; Ignacio, Caroline; Landais, Elise; Weaver, Steven; Phung, Pham; Ludka, Colleen; Hepler, Lance; Caballero, Gemma; Pollner, Tristan; Guo, Yan; Richman, Douglas; Poignard, Pascal; Paxinos, Ellen E.; Kosakovsky Pond, Sergei L.

2016-01-01

Abstract The ability to study rapidly evolving viral populations has been constrained by the read length of next-generation sequencing approaches and the sampling depth of single-genome amplification methods. Here, we develop and characterize a method using Pacific Biosciences’ Single Molecule, Real-Time (SMRT®) sequencing technology to sequence multiple, intact full-length human immunodeficiency virus-1 env genes amplified from viral RNA populations circulating in blood, and provide computational tools for analyzing and visualizing these data. PMID:29492273

Nuclear Localization of Vascular Endothelial Growth Factor-D and Regulation of c-Myc–Dependent Transcripts in Human Lung Fibroblasts

PubMed Central

Pacheco-Rodriguez, Gustavo; Malide, Daniela; Meza-Carmen, Victor; Kato, Jiro; Cui, Ye; Padilla, Philip I.; Samidurai, Arun; Gochuico, Bernadette R.

2014-01-01

Lymphangiogenesis and angiogenesis are processes that are, in part, regulated by vascular endothelial growth factor (VEGF)-D. The formation of lymphatic structures has been implicated in multiple lung diseases, including pulmonary fibrosis. VEGF-D is a secreted protein produced by fibroblasts and macrophages, which induces lymphangiogenesis by signaling via VEGF receptor-3, and angiogenesis through VEGF receptor-2. VEGF-D contains a central VEGF homology domain, which is the biologically active domain, with flanking N- and C-terminal propeptides. Full-length VEGF-D (∼ 50 kD) is proteolytically processed in the extracellular space, to generate VEGF homology domain that contains the VEGF-D receptor–binding sites. Here, we report that, independent of its cell surface receptors, full-length VEGF-D accumulated in nuclei of fibroblasts, and that this process appears to increase with cell density. In nuclei, full-length VEGF-D associated with RNA polymerase II and c-Myc. In cells depleted of VEGF-D, the transcriptionally regulated genes appear to be modulated by c-Myc. These findings have potential clinical implications, as VEGF-D was found in fibroblast nuclei in idiopathic pulmonary fibrosis, a disease characterized by fibroblast proliferation. These findings are consistent with actions of full-length VEGF-D in cellular homeostasis in health and disease, independent of its receptors. PMID:24450584

Benchmarking of the Oxford Nanopore MinION sequencing for quantitative and qualitative assessment of cDNA populations.

PubMed

Oikonomopoulos, Spyros; Wang, Yu Chang; Djambazian, Haig; Badescu, Dunarel; Ragoussis, Jiannis

2016-08-24

To assess the performance of the Oxford Nanopore Technologies MinION sequencing platform, cDNAs from the External RNA Controls Consortium (ERCC) RNA Spike-In mix were sequenced. This mix mimics mammalian mRNA species and consists of 92 polyadenylated transcripts with known concentration. cDNA libraries were generated using a template switching protocol to facilitate the direct comparison between different sequencing platforms. The MinION performance was assessed for its ability to sequence the cDNAs directly with good accuracy in terms of abundance and full length. The abundance of the ERCC cDNA molecules sequenced by MinION agreed with their expected concentration. No length or GC content bias was observed. The majority of cDNAs were sequenced as full length. Additionally, a complex cDNA population derived from a human HEK-293 cell line was sequenced on an Illumina HiSeq 2500, PacBio RS II and ONT MinION platforms. We observed that there was a good agreement in the measured cDNA abundance between PacBio RS II and ONT MinION (rpearson = 0.82, isoforms with length more than 700bp) and between Illumina HiSeq 2500 and ONT MinION (rpearson = 0.75). This indicates that the ONT MinION can sequence quantitatively both long and short full length cDNA molecules.

A baculovirus-mediated strategy for full-length plant virus coat protein expression and purification.

PubMed

Ardisson-Araújo, Daniel Mendes Pereira; Rocha, Juliana Ribeiro; da Costa, Márcio Hedil Oliveira; Bocca, Anamélia Lorenzetti; Dusi, André Nepomuceno; de Oliveira Resende, Renato; Ribeiro, Bergmann Morais

2013-08-15

Garlic production is severely affected by virus infection, causing a decrease in productivity and quality. There are no virus-free cultivars and garlic-infecting viruses are difficult to purify, which make specific antibody production very laborious. Since high quality antisera against plant viruses are important tools for serological detection, we have developed a method to express and purify full-length plant virus coat proteins using baculovirus expression system and insects as bioreactors. In this work, we have fused the full-length coat protein (cp) gene from the Garlic Mite-borne Filamentous Virus (GarMbFV) to the 3'-end of the Polyhedrin (polh) gene of the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV). The recombinant baculovirus was amplified in insect cell culture and the virus was used to infect Spodoptera frugiperda larvae. Thus, the recombinant fused protein was easily purified from insect cadavers using sucrose gradient centrifugation and analyzed by Western Blotting. Interestingly, amorphous crystals were produced in the cytoplasm of cells infected with the recombinant virus containing the chimeric-protein gene but not in cells infected with the wild type and recombinant virus containing the hexa histidine tagged Polh. Moreover, the chimeric protein was used to immunize rats and generate antibodies against the target protein. The antiserum produced was able to detect plants infected with GarMbFV, which had been initially confirmed by RT-PCR. The expression of a plant virus full-length coat protein fused to the baculovirus Polyhedrin in recombinant baculovirus-infected insects was shown to produce high amounts of the recombinant protein which was easily purified and efficiently used to generate specific antibodies. Therefore, this strategy can potentially be used for the development of plant virus diagnostic kits for those viruses that are difficult to purify, are present in low titers or are present in mix infection in their plant hosts.

TypeLoader: A fast and efficient automated workflow for the annotation and submission of novel full-length HLA alleles.

PubMed

Surendranath, V; Albrecht, V; Hayhurst, J D; Schöne, B; Robinson, J; Marsh, S G E; Schmidt, A H; Lange, V

2017-07-01

Recent years have seen a rapid increase in the discovery of novel allelic variants of the human leukocyte antigen (HLA) genes. Commonly, only the exons encoding the peptide binding domains of novel HLA alleles are submitted. As a result, the IPD-IMGT/HLA Database lacks sequence information outside those regions for the majority of known alleles. This has implications for the application of the new sequencing technologies, which deliver sequence data often covering the complete gene. As these technologies simplify the characterization of the complete gene regions, it is desirable for novel alleles to be submitted as full-length sequences to the database. However, the manual annotation of full-length alleles and the generation of specific formats required by the sequence repositories is prone to error and time consuming. We have developed TypeLoader to address both these facets. With only the full-length sequence as a starting point, Typeloader performs automatic sequence annotation and subsequently handles all steps involved in preparing the specific formats for submission with very little manual intervention. TypeLoader is routinely used at the DKMS Life Science Lab and has aided in the successful submission of more than 900 novel HLA alleles as full-length sequences to the European Nucleotide Archive repository and the IPD-IMGT/HLA Database with a 95% reduction in the time spent on annotation and submission when compared with handling these processes manually. TypeLoader is implemented as a web application and can be easily installed and used on a standalone Linux desktop system or within a Linux client/server architecture. TypeLoader is downloadable from http://www.github.com/DKMS-LSL/typeloader. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Pseudo-polyprotein translated from the full-length ORF1 of capillovirus is important for pathogenicity, but a truncated ORF1 protein without variable and CP regions is sufficient for replication.

PubMed

Hirata, Hisae; Yamaji, Yasuyuki; Komatsu, Ken; Kagiwada, Satoshi; Oshima, Kenro; Okano, Yukari; Takahashi, Shuichiro; Ugaki, Masashi; Namba, Shigetou

2010-09-01

The first open-reading frame (ORF) of the genus Capillovirus encodes an apparently chimeric polyprotein containing conserved regions for replicase (Rep) and coat protein (CP), while other viruses in the family Flexiviridae have separate ORFs encoding these proteins. To investigate the role of the full-length ORF1 polyprotein of capillovirus, we generated truncation mutants of ORF1 of apple stem grooving virus by inserting a termination codon into the variable region located between the putative Rep- and CP-coding regions. These mutants were capable of systemic infection, although their pathogenicity was attenuated. In vitro translation of ORF1 produced both the full-length polyprotein and the smaller Rep protein. The results of in vivo reporter assays suggested that the mechanism of this early termination is a ribosomal -1 frame-shift occurring downstream from the conserved Rep domains. The mechanism of capillovirus gene expression and the very close evolutionary relationship between the genera Capillovirus and Trichovirus are discussed. Copyright (c) 2010. Published by Elsevier B.V.

Production of functional human insulin-like growth factor binding proteins (IGFBPs) using recombinant expression in HEK293 cells.

PubMed

Wanscher, Anne Sofie Molsted; Williamson, Michael; Ebersole, Tasja Wainani; Streicher, Werner; Wikström, Mats; Cazzamali, Giuseppe

2015-04-01

Insulin-like growth factor binding proteins (IGFBPs) display many functions in humans including regulation of the insulin-like growth factor (IGF) signaling pathway. The various roles of human IGFBPs make them attractive protein candidates in drug discovery. Structural and functional knowledge on human proteins with therapeutic relevance is needed to design and process the next generation of protein therapeutics. In order to conduct structural and functional investigations large quantities of recombinant proteins are needed. However, finding a suitable recombinant production system for proteins such as full-length human IGFBPs, still remains a challenge. Here we present a mammalian HEK293 expression method suitable for over-expression of secretory full-length human IGFBP-1 to -7. Protein purification of full-length human IGFBP-1, -2, -3 and -5 was conducted using a two-step chromatography procedure and the final protein yields were between 1 and 12mg protein per liter culture media. The recombinant IGFBPs contained PTMs and exhibited high-affinity interactions with their natural ligands IGF-1 and IGF-2. Copyright © 2014 Elsevier Inc. All rights reserved.

Hormone Binding to Recombinant Estrogen Receptors from Human, Alligator, Quail, Salamander, and Fathead Minnow

EPA Science Inventory

In this work, a 96-well plate estrogen receptor binding assay was developed to facilitate the direct comparison of chemical binding to full-length recombinant estrogen receptors across vertebrate classes. Receptors were generated in a baculovirus expression system. This approach ...

FOX-superroots of Lotus corniculatus, overexpressing Arabidopsis full-length cDNA, show stable variations in morphological traits.

PubMed

Himuro, Yasuyo; Tanaka, Hidenori; Hashiguchi, Masatsugu; Ichikawa, Takanari; Nakazawa, Miki; Seki, Motoaki; Fujita, Miki; Shinozaki, Kazuo; Matsui, Minami; Akashi, Ryo; Hoffmann, Franz

2011-01-15

Using the full-length cDNA overexpressor (FOX) gene-hunting system, we have generated 130 Arabidopsis FOX-superroot lines in bird's-foot trefoil (Lotus corniculatus) for the systematic functional analysis of genes expressed in roots and for the selection of induced mutants with interesting root growth characteristics. We used the Arabidopsis-FOX Agrobacterium library (constructed by ligating pBIG2113SF) for the Agrobacterium-mediated transformation of superroots (SR) and the subsequent selection of gain-of-function mutants with ectopically expressed Arabidopsis genes. The original superroot culture of L. corniculatus is a unique host system displaying fast root growth in vitro, allowing continuous root cloning, direct somatic embryogenesis and mass regeneration of plants under entirely hormone-free culture conditions. Several of the Arabidopsis FOX-superroot lines show interesting deviations from normal growth and morphology of roots from SR-plants, such as differences in pigmentation, growth rate, length or diameter. Some of these mutations are of potential agricultural interest. Genomic PCR analysis revealed that 100 (76.9%) out of the 130 transgenic lines showed the amplification of single fragments. Sequence analysis of the PCR fragments from these 100 lines identified full-length cDNA in 74 of them. Forty-three out of 74 full-length cDNA carried known genes. The Arabidopsis FOX-superroot lines of L. corniculatus, produced in this study, expand the FOX hunting system and provide a new tool for the genetic analysis and control of root growth in a leguminous forage plant. Copyright © 2010 Elsevier GmbH. All rights reserved.

California mild CTV strains that break resistance in Trifoliate Orange

USDA-ARS?s Scientific Manuscript database

This is the final report of a project to characterize California isolates of Citrus tristeza virus (CTV) that replicate in Poncirus trifoliata (trifoliate orange). Next Generation Sequencing (NGS) of viral small interfering RNAs (siRNAs) and assembly of full-length sequences of mild California CTV i...

NASA-STD-6001B Test 1 Upward Flame Propagation; Sample Length Impact on MOC Investigation

NASA Technical Reports Server (NTRS)

Harper, Susana Tapia; Juarez, Alfredo; Woods, Brenton L.; Beeson, Harold D.

2017-01-01

Understanding the combustion behavior of materials in the elevated oxygen environments of habitable spacecraft is of utmost importance to crew safety and mission success. Currently, certification for unrestricted flight usage of a material with respect to flammability involves passing the Upward Flame Propagation Test of NASA-STD-6001B (Test 1). This test evaluates materials in a standardized test configuration for two failure criteria: self-extinguishment within 15 cm (6 in.) and the propensity of flame propagation by means of flaming material transfer. By the NASA standard, full-length samples are 30 cm (12 in.) in length; however, factors independent of the test method such as limited material availability or various nonstandard test configurations limit the full pretest sample lengths available for test. This paper characterizes the dependence, if any, of pretest sample length on NASA-STD-6001B Test 1 results. Testing was performed using the Maximum Oxygen Concentration (MOC) Threshold Method to obtain a data set for each sample length tested. In addition, various material types, including cloth (Nomex), foam (TA-301) and solids (Ultem), were tested to investigate potential effects of test specimen types. Though additional data needs to be generated to provide statistical confidence, preliminary findings are that use of variable sample lengths has minimal impact on NASA-STD-6001B flammability performance and MOC determination.

A Hybrid Approach for the Automated Finishing of Bacterial Genomes

PubMed Central

Robins, William P.; Chin, Chen-Shan; Webster, Dale; Paxinos, Ellen; Hsu, David; Ashby, Meredith; Wang, Susana; Peluso, Paul; Sebra, Robert; Sorenson, Jon; Bullard, James; Yen, Jackie; Valdovino, Marie; Mollova, Emilia; Luong, Khai; Lin, Steven; LaMay, Brianna; Joshi, Amruta; Rowe, Lori; Frace, Michael; Tarr, Cheryl L.; Turnsek, Maryann; Davis, Brigid M; Kasarskis, Andrew; Mekalanos, John J.; Waldor, Matthew K.; Schadt, Eric E.

2013-01-01

Dramatic improvements in DNA sequencing technology have revolutionized our ability to characterize most genomic diversity. However, accurate resolution of large structural events has remained challenging due to the comparatively shorter read lengths of second-generation technologies. Emerging third-generation sequencing technologies, which yield markedly increased read length on rapid time scales and for low cost, have the potential to address assembly limitations. Here we combine sequencing data from second- and third-generation DNA sequencing technologies to assemble the two-chromosome genome of a recent Haitian cholera outbreak strain into two nearly finished contigs at > 99.9% accuracy. Complex regions with clinically significant structure were completely resolved. In separate control assemblies on experimental and simulated data for the canonical N16961 reference we obtain 14 and 8 scaffolds greater than 1kb, respectively, correcting several errors in the underlying source data. This work provides a blueprint for the next generation of rapid microbial identification and full-genome assembly. PMID:22750883

Post-translational generation of constitutively active cores from larger phosphatases in the malaria parasite, Plasmodium falciparum: implications for proteomics

PubMed Central

Kumar, Rajinder; Musiyenko, Alla; Oldenburg, Anja; Adams, Brian; Barik, Sailen

2004-01-01

Background Although the complete genome sequences of a large number of organisms have been determined, the exact proteomes need to be characterized. More specifically, the extent to which post-translational processes such as proteolysis affect the synthesized proteins has remained unappreciated. We examined this issue in selected protein phosphatases of the protease-rich malaria parasite, Plasmodium falciparum. Results P. falciparum encodes a number of Ser/Thr protein phosphatases (PP) whose catalytic subunits are composed of a catalytic core and accessory domains essential for regulation of the catalytic activity. Two examples of such regulatory domains are found in the Ca+2-regulated phosphatases, PP7 and PP2B (calcineurin). The EF-hand domains of PP7 and the calmodulin-binding domain of PP2B are essential for stimulation of the phosphatase activity by Ca+2. We present biochemical evidence that P. falciparum generates these full-length phosphatases as well as their catalytic cores, most likely as intermediates of a proteolytic degradation pathway. While the full-length phosphatases are activated by Ca+2, the processed cores are constitutively active and either less responsive or unresponsive to Ca+2. The processing is extremely rapid, specific, and occurs in vivo. Conclusions Post-translational cleavage efficiently degrades complex full-length phosphatases in P. falciparum. In the course of such degradation, enzymatically active catalytic cores are produced as relatively stable intermediates. The universality of such proteolysis in other phosphatases or other multi-domain proteins and its potential impact on the overall proteome of a cell merits further investigation. PMID:15230980

Enhancing the Breadth and Efficacy of Therapeutic Vaccines for Breast Cancer

DTIC Science & Technology

2014-10-01

us to generate full length recombinant TCR proteins that will be used to screen against the bacteriophage peptide library the Slansky/Kappler team has...the Denver team bacteriophage peptide library for HLA-A2 to screen these T cells. As a result we have abandoned this approach in order to focus our

Improved Synthesis and In Vitro Evaluation of an Aptamer Ribosomal Toxin Conjugate

PubMed Central

Kelly, Linsley; Kratschmer, Christina; Maier, Keith E.; Yan, Amy C.

2016-01-01

Delivery of toxins, such as the ricin A chain, Pseudomonas exotoxin, and gelonin, using antibodies has had some success in inducing specific toxicity in cancer treatments. However, these antibody-toxin conjugates, called immunotoxins, can be bulky, difficult to express, and may induce an immune response upon in vivo administration. We previously reported delivery of a recombinant variant of gelonin (rGel) by the full-length prostate-specific membrane antigen (PSMA) binding aptamer, A9, to potentially circumvent some of these problems. Here, we report a streamlined approach to generating aptamer-rGel conjugates utilizing a chemically synthesized minimized form of the A9 aptamer. Unlike the full-length A9 aptamer, this minimized variant can be chemically synthesized with a 5′ terminal thiol. This facilitates the large scale synthesis and generation of aptamer toxin conjugates linked by a reducible disulfide linkage. Using this approach, we generated aptamer-toxin conjugates and evaluated their binding specificity and toxicity. On PSMA(+) LNCaP prostate cancer cells, the A9.min-rGel conjugate demonstrated an IC50 of ∼60 nM. Additionally, we performed a stability analysis of this conjugate in mouse serum where the conjugate displayed a t1/2 of ∼4 h, paving the way for future in vivo experiments. PMID:27228412

Near full-length 16S rRNA gene next-generation sequencing revealed Asaia as a common midgut bacterium of wild and domesticated Queensland fruit fly larvae.

PubMed

Deutscher, Ania T; Burke, Catherine M; Darling, Aaron E; Riegler, Markus; Reynolds, Olivia L; Chapman, Toni A

2018-05-05

Gut microbiota affects tephritid (Diptera: Tephritidae) fruit fly development, physiology, behavior, and thus the quality of flies mass-reared for the sterile insect technique (SIT), a target-specific, sustainable, environmentally benign form of pest management. The Queensland fruit fly, Bactrocera tryoni (Tephritidae), is a significant horticultural pest in Australia and can be managed with SIT. Little is known about the impacts that laboratory-adaptation (domestication) and mass-rearing have on the tephritid larval gut microbiome. Read lengths of previous fruit fly next-generation sequencing (NGS) studies have limited the resolution of microbiome studies, and the diversity within populations is often overlooked. In this study, we used a new near full-length (> 1300 nt) 16S rRNA gene amplicon NGS approach to characterize gut bacterial communities of individual B. tryoni larvae from two field populations (developing in peaches) and three domesticated populations (mass- or laboratory-reared on artificial diets). Near full-length 16S rRNA gene sequences were obtained for 56 B. tryoni larvae. OTU clustering at 99% similarity revealed that gut bacterial diversity was low and significantly lower in domesticated larvae. Bacteria commonly associated with fruit (Acetobacteraceae, Enterobacteriaceae, and Leuconostocaceae) were detected in wild larvae, but were largely absent from domesticated larvae. However, Asaia, an acetic acid bacterium not frequently detected within adult tephritid species, was detected in larvae of both wild and domesticated populations (55 out of 56 larval gut samples). Larvae from the same single peach shared a similar gut bacterial profile, whereas larvae from different peaches collected from the same tree had different gut bacterial profiles. Clustering of the Asaia near full-length sequences at 100% similarity showed that the wild flies from different locations had different Asaia strains. Variation in the gut bacterial communities of B. tryoni larvae depends on diet, domestication, and horizontal acquisition. Bacterial variation in wild larvae suggests that more than one bacterial species can perform the same functional role; however, Asaia could be an important gut bacterium in larvae and warrants further study. A greater understanding of the functions of the bacteria detected in larvae could lead to increased fly quality and performance as part of the SIT.

Therapeutic footwear: enhanced function in people with diabetes and transmetatarsal amputation.

PubMed

Mueller, M J; Strube, M J

1997-09-01

Patients with diabetes mellitus (DM) and a transmetatarsal amputation (TMA) have considerable deficits in function compared with age-matched controls. The purpose of this study was to determine if therapeutic footwear could improve the functional mobility of patients with DM and TMA. Repeated-measures design. Academic medical center. Thirty subjects (10 women, 20 men) with DM and a TMA, with a mean age of 61.7 +/- 4.0 yrs. Six types of footwear evaluating the following components: length of shoe (full-length or short shoe), a rigid rocker-bottom sole, and an ankle-foot-orthosis. Physical Performance Test (PPT), functional reach, and walking speed. Measurements in each footwear condition occurred after a 1-month adjustment period. Patients wearing full-length custom-made shoes with a total-contact insert, a rigid rocker-bottom sole or a short shoe with a rigid rocker-bottom sole (with or without an ankle-foot-orthosis) had similar and significantly higher scores in the PPT and faster walking speed than when wearing regular shoes with a toe filler (p < .05). The short shoe and the ankle-foot-orthosis, however, generated many patient complaints about cosmesis and restriction at the ankle, respectively. There were no differences in any of the measures of functional reach. Although there are individual exceptions, we recommend the full-length shoe, total-contact insert, and a rigid rocker-bottom sole for most patients with DM and a TMA.

Complementary DNA libraries: an overview.

PubMed

Ying, Shao-Yao

2004-07-01

The generation of complete and full-length cDNA libraries for potential functional assays of specific gene sequences is essential for most molecules in biotechnology and biomedical research. The field of cDNA library generation has changed rapidly in the past 10 yr. This review presents an overview of the method available for the basic information of generating cDNA libraries, including the definition of the cDNA library, different kinds of cDNA libraries, difference between methods for cDNA library generation using conventional approaches and a novel strategy, and the quality of cDNA libraries. It is anticipated that the high-quality cDNA libraries so generated would facilitate studies involving genechips and the microarray, differential display, subtractive hybridization, gene cloning, and peptide library generation.

In vitro transcripts of wild-type and fluorescent protein-tagged triticum mosaic virus (family potyviridae) are biologically active in wheat

USDA-ARS?s Scientific Manuscript database

Triticum mosaic virus (TriMV) (genus Poacevirus, family Potyviridae) is a recently described eriophyid mite-transmitted wheat virus. In vitro RNA transcripts generated from full-length cDNA clones of TriMV proved infectious on wheat, and the progeny virus was efficiently transmitted by wheat curl m...

Design and construction of a first-generation high-throughput integrated robotic molecular biology platform for bioenergy applications

USDA-ARS?s Scientific Manuscript database

The molecular biological techniques for plasmid-based assembly and cloning of gene open reading frames are essential for elucidating the function of the proteins encoded by the genes. These techniques involve the production of full-length cDNA libraries as a source of plasmid-based clones to expres...

PCR Amplification Strategies towards full-length HIV-1 Genome sequencing.

PubMed

Liu, Chao Chun; Ji, Hezhao

2018-06-26

The advent of next generation sequencing has enabled greater resolution of viral diversity and improved feasibility of full viral genome sequencing allowing routine HIV-1 full genome sequencing in both research and diagnostic settings. Regardless of the sequencing platform selected, successful PCR amplification of the HIV-1 genome is essential for sequencing template preparation. As such, full HIV-1 genome amplification is a crucial step in dictating the successful and reliable sequencing downstream. Here we reviewed existing PCR protocols leading to HIV-1 full genome sequencing. In addition to the discussion on basic considerations on relevant PCR design, the advantages as well as the pitfalls of published protocols were reviewed. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

High-brightness diode pump sources for solid-state and fiber laser pumping across 8xx-9xx nm range

NASA Astrophysics Data System (ADS)

Diamant, Ronen; Berk, Yuri; Cohen, Shalom; Klumel, Genady; Levy, Moshe; Openhaim, Yaki; Peleg, Ophir; Yanson, Dan; Karni, Yoram

2011-06-01

Advanced solid state laser architectures place increasingly demanding requirements on high-brightness, low-cost QCW laser diode pump sources, with custom apertures both for side and end rod pumping configurations. To meet this need, a new series of scalable QCW pump sources at 808nm and 940nm was developed. The stacks, available in multiple output formats, allow for custom aperture filling by varying both the length and quantity of stacked laser bars. For these products, we developed next-generation laser bars based on improved epitaxial wafer designs delivering power densities of 20W/mm of emission aperture. With >200W of peak QCW power available from a full-length 1cm bar, we have demonstrated power scaling to over 2kW in 10-bar stacks with 55% wall plug efficiency. We also present the design and performance of several stack configurations using full-length and reduced-length (mini) bars that demonstrate the versatility of both the bar and packaging designs. We illustrate how the ROBUST HEAD packaging technology developed at SCD is capable of accommodating variable bar length, pitch and quantity for custom rod pumping geometries. The excellent all-around performance of the stacks is supported by reliability data in line with the previously reported 20 Gshot space-grade qualification of SCD's stacks.

Scaleable multi-format QCW pump stacks based on 200W laser diode bars and mini bars at 808nm and 940nm

NASA Astrophysics Data System (ADS)

Berk, Yuri; Karni, Yoram; Klumel, Genady; Openhaim, Yaakov; Cohen, Shalom; Yanson, Dan

2011-03-01

Advanced solid state laser architectures place increasingly demanding requirements on high-brightness, low-cost QCW laser diode pump sources, with custom apertures both for side and end rod pumping configurations. To meet this need, a new series of scaleable pump sources at 808nm and 940nm was developed. The stacks, available in multiple output formats, allow for custom aperture filling by varying both the length and quantity of stacked laser bars. For these products, we developed next-generation laser bars based on improved epitaxial wafer designs delivering power densities of 20W/mm of emission aperture. With >200W of peak QCW power available from a full-length 1cm bar, we have demonstrated power scaling to over 2kW in 10-bar stacks with 55% wall plug efficiency. We also present the design and performance of several stack configurations using full-length and reduced-length (mini) bars that demonstrate the versatility of both the bar and packaging designs. We illustrate how the ROBUST HEAD packaging technology developed at SCD is capable of accommodating variable bar length, pitch and quantity for custom rod pumping geometries. The excellent all-around performance of the stacks is supported by reliability data in line with the previously reported 20 Gshot space-grade qualification of SCD's stacks.

Metagenomic and near full-length 16S rRNA sequence data in support of the phylogenetic analysis of the rumen bacterial community in steers

USDA-ARS?s Scientific Manuscript database

Next generation sequencing technologies have vastly changed the approach of sequencing of the 16S rRNA gene for studies in microbial ecology. Three distinct technologies are available for large-scale 16S sequencing. All three are subject to biases introduced by sequencing error rates, amplificatio...

Design and construction of a first-generation high-throughput integrated molecular biology platform for production of optimized synthetic genes and improved industrial strains

USDA-ARS?s Scientific Manuscript database

The molecular biological techniques for plasmid-based assembly and cloning of synthetic assembled gene open reading frames are essential for elucidating the function of the proteins encoded by the genes. These techniques involve the production of full-length cDNA libraries as a source of plasmid-bas...

78 FR 13071 - Guidance for Industry: Implementation of an Acceptable Full-Length and Abbreviated Donor History...

Federal Register 2010, 2011, 2012, 2013, 2014

2013-02-26

...] Guidance for Industry: Implementation of an Acceptable Full- Length and Abbreviated Donor History... Full-Length and Abbreviated Donor History Questionnaires and Accompanying Materials for Use in... full-length and abbreviated donor history questionnaires and accompanying materials, version 1.2 dated...

Knee and Ankle Joint Angles Influence the Plantarflexion Torque of the Gastrocnemius.

PubMed

Landin, Dennis; Thompson, Melissa; Reid, Meghan

2015-08-01

The gastrocnemius (GA) is the lone bi-articular muscle of the leg, crossing both the knee and ankle. As with any bi-articular muscle, both joints affect its length/tension curve. The role of the GA as a plantarflexor is firmly established; however, no current research has investigated how changes in knee and ankle joint positions on its ability to generate a plantarflexion (PF) torque. This paper reports on the PF force generated by the GA at specific knee and ankle joint combinations. The right GA of 26 participants was electrically stimulated via surface electrodes following a standardized protocol at 24 knee and ankle joint combinations. Three stimulations were applied at each of the 24 positions. Data were recorded on three dependent measures: the passive moment, which was the PF moment created by the tissue without stimulation, the maximum moment, which was the highest PF moment during the stimulation and included the passive moment, and the stimulated moment, which reflected the PF moment during stimulation minus the passive moment. A straight knee and dorsiflexed ankle create the position in which the GA generates the greatest PF moment, but it is also the position of greatest length. This finding is in contrast to conclusions from previous research with bi-articular muscles, which has consistently shown that the greatest length is not a muscle's optimal length. The full ranges of motion for the knee and ankle apparently do not elongate the GA beyond its optimal length for producing a PF moment. Clinicians commonly evaluate GA status with the patient seated and the foot subject to gravity. The present results indicate that manual testing of the GA in isolation should be performed, whenever possible, with the knee extended and the ankle dorsiflexed to potentially elicit the maximum PF torque from the GA.

ZResponse to selection, heritability and genetic correlations between body weight and body size in Pacific white shrimp, Litopenaeus vannamei

NASA Astrophysics Data System (ADS)

Andriantahina, Farafidy; Liu, Xiaolin; Huang, Hao; Xiang, Jianhai

2012-03-01

To quantify the response to selection, heritability and genetic correlations between weight and size of Litopenaeus vannamei, the body weight (BW), total length (TL), body length (BL), first abdominal segment depth (FASD), third abdominal segment depth (TASD), first abdominal segment width (FASW), and partial carapace length (PCL) of 5-month-old parents and of offspnng were measured by calculating seven body measunngs of offspnng produced by a nested mating design. Seventeen half-sib families and 42 full-sib families of L. vannamei were produced using artificial fertilization from 2-4 dams by each sire, and measured at around five months post-metamorphosis. The results show that hentabilities among vanous traits were high: 0.515±0.030 for body weight and 0.394±0.030 for total length. After one generation of selection. the selection response was 10.70% for offspring growth. In the 5th month, the realized heritability for weight was 0.296 for the offspnng generation. Genetic correlations between body weight and body size were highly variable. The results indicate that external morphological parameters can be applied dunng breeder selection for enhancing the growth without sacrificing animals for determining the body size and breed ability; and selective breeding can be improved significantly, simultaneously with increased production.

Rapid Construction of Stable Infectious Full-Length cDNA Clone of Papaya Leaf Distortion Mosaic Virus Using In-Fusion Cloning

PubMed Central

Tuo, Decai; Shen, Wentao; Yan, Pu; Li, Xiaoying; Zhou, Peng

2015-01-01

Papaya leaf distortion mosaic virus (PLDMV) is becoming a threat to papaya and transgenic papaya resistant to the related pathogen, papaya ringspot virus (PRSV). The generation of infectious viral clones is an essential step for reverse-genetics studies of viral gene function and cross-protection. In this study, a sequence- and ligation-independent cloning system, the In-Fusion® Cloning Kit (Clontech, Mountain View, CA, USA), was used to construct intron-less or intron-containing full-length cDNA clones of the isolate PLDMV-DF, with the simultaneous scarless assembly of multiple viral and intron fragments into a plasmid vector in a single reaction. The intron-containing full-length cDNA clone of PLDMV-DF was stably propagated in Escherichia coli. In vitro intron-containing transcripts were processed and spliced into biologically active intron-less transcripts following mechanical inoculation and then initiated systemic infections in Carica papaya L. seedlings, which developed similar symptoms to those caused by the wild-type virus. However, no infectivity was detected when the plants were inoculated with RNA transcripts from the intron-less construct because the instability of the viral cDNA clone in bacterial cells caused a non-sense or deletion mutation of the genomic sequence of PLDMV-DF. To our knowledge, this is the first report of the construction of an infectious full-length cDNA clone of PLDMV and the splicing of intron-containing transcripts following mechanical inoculation. In-Fusion cloning shortens the construction time from months to days. Therefore, it is a faster, more flexible, and more efficient method than the traditional multistep restriction enzyme-mediated subcloning procedure. PMID:26633465

A Short Isoform of Human Cytomegalovirus US3 Functions as a Dominant Negative Inhibitor of the Full-Length Form

PubMed Central

Shin, Jinwook; Park, Boyoun; Lee, Sungwook; Kim, Youngkyun; Biegalke, Bonita J.; Kang, Seongman; Ahn, Kwangseog

2006-01-01

Human cytomegalovirus encodes four unique short (US) region proteins, each of which is independently sufficient for causing the down-regulation of major histocompatibility complex (MHC) class I molecules on the cell surface. This down-regulation enables infected cells to evade recognition by cytotoxic T lymphocytes (CTLs) but makes them vulnerable to lysis by natural killer (NK) cells, which lyse those cells that lack MHC class I molecules. The 22-kDa US3 glycoprotein is able to down-regulate the surface expression of MHC class I molecules by dual mechanisms: direct endoplasmic reticulum retention by physical association and/or tapasin inhibition. The alternative splicing of the US3 gene generates two additional products, including 17-kDa and 3.5-kDa truncated isoforms; however, the functional significance of these isoforms during viral infection is unknown. Here, we describe a novel mode of self-regulation of US3 function that uses the endogenously produced truncated isoform. The truncated isoform itself neither binds to MHC class I molecules nor prevents the full-length US3 from interacting with MHC class I molecules. Instead, the truncated isoform associates with tapasin and competes with full-length US3 for binding to tapasin; thus, it suppresses the action of US3 that causes the disruption of the function of tapasin. Our results indicate that the truncated isoform of the US3 locus acts as a dominant negative regulator of full-length US3 activity. These data reflect the manner in which the virus has developed temporal survival strategies during viral infection against immune surveillance involving both CTLs and NK cells. PMID:16699020

Deep sequencing of near full-length HIV-1 genomes from plasma identifies circulating subtype C and infrequent occurrence of AC recombinant form in Southern India

PubMed Central

Sampathkumar, Raghavan; Sivaraman, Karthi; U. K. J., Anto Jesuraj; Dhar, Chirag; D. Souza, George; Berry, Neil

2017-01-01

India has the third largest number of HIV-1-infected individuals accounting for approximately 2.1 million people, with a predominance of circulating subtype C strains and a low prevalence of subtype A and A1C and BC recombinant forms, identified over the past two decades. Recovery of near full-length HIV-1 genomes from a plasma source coupled with advances in next generation sequencing (NGS) technologies and development of universal methods for amplifying whole genomes of HIV-1 circulating in a target geography or population provides the opportunity for a detailed analysis of HIV-1 strain identification, evolution and dynamics. Here we describe the development and implementation of approaches for HIV-1 NGS analysis in a southern Indian cohort. Plasma samples (n = 20) were obtained from HIV-1-confirmed individuals living in and around the city of Bengaluru. Near full-length genome recovery was obtained for 9 Indian HIV-1 patients, with recovery of full-length gag and env genes for 10 and 2 additional subjects, respectively. Phylogenetic analyses indicate the majority of sequences to be represented by subtype C viruses branching within a monophyletic clade, comprising viruses from India, Nepal, Myanmar and China and closely related to a southern African cluster, with a low prevalence of the A1C recombinant form also present. Development of algorithms for bespoke recovery and analysis at a local level will further aid clinical management of HIV-1 infected Indian subjects and delineate the progress of the HIV-1 pandemic in this and other geographical regions. PMID:29220350

Functional PAK-2 knockout and replacement with a caspase cleavage-deficient mutant in mice reveals differential requirements of full-length PAK-2 and caspase-activated PAK-2p34.

PubMed

Marlin, Jerry W; Chang, Yu-Wen E; Ober, Margaret; Handy, Amy; Xu, Wenhao; Jakobi, Rolf

2011-06-01

p21-Activated protein kinase 2 (PAK-2) has both anti- and pro-apoptotic functions depending on its mechanism of activation. Activation of full-length PAK-2 by the monomeric GTPases Cdc42 or Rac stimulates cell survival, whereas caspase activation of PAK-2 to the PAK-2p34 fragment is involved in the apoptotic response. In this study we use functional knockout of PAK-2 and gene replacement with the caspase cleavage-deficient PAK-2D212N mutant to differentiate the biological functions of full-length PAK-2 and caspase-activated PAK-2p34. Knockout of PAK-2 results in embryonic lethality at early stages before organ development, whereas replacement with the caspase cleavage-deficient PAK-2D212N results in viable and healthy mice, indicating that early embryonic lethality is caused by deficiency of full-length PAK-2 rather than lack of caspase activation to the PAK-2p34 fragment. However, deficiency of caspase activation of PAK-2 decreased spontaneous cell death of primary mouse embryonic fibroblasts and increased cell growth at high cell density. In contrast, stress-induced cell death by treatment with the anti-cancer drug cisplatin was not reduced by deficiency of caspase activation of PAK-2, but switched from an apoptotic to a nonapoptotic, caspase-independent mechanism. Homozygous PAK-2D212N primary mouse embryonic fibroblasts that lack the ability to generate the proapoptotic PAK-2p34 show less activation of the effector caspase 3, 6, and 7, indicating that caspase activation of PAK-2 amplifies the apoptotic response through a positive feedback loop resulting in more activation of effector caspases.

Rapid Construction of Stable Infectious Full-Length cDNA Clone of Papaya Leaf Distortion Mosaic Virus Using In-Fusion Cloning.

PubMed

Tuo, Decai; Shen, Wentao; Yan, Pu; Li, Xiaoying; Zhou, Peng

2015-12-01

Papaya leaf distortion mosaic virus (PLDMV) is becoming a threat to papaya and transgenic papaya resistant to the related pathogen, papaya ringspot virus (PRSV). The generation of infectious viral clones is an essential step for reverse-genetics studies of viral gene function and cross-protection. In this study, a sequence- and ligation-independent cloning system, the In-Fusion(®) Cloning Kit (Clontech, Mountain View, CA, USA), was used to construct intron-less or intron-containing full-length cDNA clones of the isolate PLDMV-DF, with the simultaneous scarless assembly of multiple viral and intron fragments into a plasmid vector in a single reaction. The intron-containing full-length cDNA clone of PLDMV-DF was stably propagated in Escherichia coli. In vitro intron-containing transcripts were processed and spliced into biologically active intron-less transcripts following mechanical inoculation and then initiated systemic infections in Carica papaya L. seedlings, which developed similar symptoms to those caused by the wild-type virus. However, no infectivity was detected when the plants were inoculated with RNA transcripts from the intron-less construct because the instability of the viral cDNA clone in bacterial cells caused a non-sense or deletion mutation of the genomic sequence of PLDMV-DF. To our knowledge, this is the first report of the construction of an infectious full-length cDNA clone of PLDMV and the splicing of intron-containing transcripts following mechanical inoculation. In-Fusion cloning shortens the construction time from months to days. Therefore, it is a faster, more flexible, and more efficient method than the traditional multistep restriction enzyme-mediated subcloning procedure.

A short isoform of human cytomegalovirus US3 functions as a dominant negative inhibitor of the full-length form.

PubMed

Shin, Jinwook; Park, Boyoun; Lee, Sungwook; Kim, Youngkyun; Biegalke, Bonita J; Kang, Seongman; Ahn, Kwangseog

2006-06-01

Human cytomegalovirus encodes four unique short (US) region proteins, each of which is independently sufficient for causing the down-regulation of major histocompatibility complex (MHC) class I molecules on the cell surface. This down-regulation enables infected cells to evade recognition by cytotoxic T lymphocytes (CTLs) but makes them vulnerable to lysis by natural killer (NK) cells, which lyse those cells that lack MHC class I molecules. The 22-kDa US3 glycoprotein is able to down-regulate the surface expression of MHC class I molecules by dual mechanisms: direct endoplasmic reticulum retention by physical association and/or tapasin inhibition. The alternative splicing of the US3 gene generates two additional products, including 17-kDa and 3.5-kDa truncated isoforms; however, the functional significance of these isoforms during viral infection is unknown. Here, we describe a novel mode of self-regulation of US3 function that uses the endogenously produced truncated isoform. The truncated isoform itself neither binds to MHC class I molecules nor prevents the full-length US3 from interacting with MHC class I molecules. Instead, the truncated isoform associates with tapasin and competes with full-length US3 for binding to tapasin; thus, it suppresses the action of US3 that causes the disruption of the function of tapasin. Our results indicate that the truncated isoform of the US3 locus acts as a dominant negative regulator of full-length US3 activity. These data reflect the manner in which the virus has developed temporal survival strategies during viral infection against immune surveillance involving both CTLs and NK cells.

Large-scale analysis of full-length cDNAs from the tomato (Solanum lycopersicum) cultivar Micro-Tom, a reference system for the Solanaceae genomics.

PubMed

Aoki, Koh; Yano, Kentaro; Suzuki, Ayako; Kawamura, Shingo; Sakurai, Nozomu; Suda, Kunihiro; Kurabayashi, Atsushi; Suzuki, Tatsuya; Tsugane, Taneaki; Watanabe, Manabu; Ooga, Kazuhide; Torii, Maiko; Narita, Takanori; Shin-I, Tadasu; Kohara, Yuji; Yamamoto, Naoki; Takahashi, Hideki; Watanabe, Yuichiro; Egusa, Mayumi; Kodama, Motoichiro; Ichinose, Yuki; Kikuchi, Mari; Fukushima, Sumire; Okabe, Akiko; Arie, Tsutomu; Sato, Yuko; Yazawa, Katsumi; Satoh, Shinobu; Omura, Toshikazu; Ezura, Hiroshi; Shibata, Daisuke

2010-03-30

The Solanaceae family includes several economically important vegetable crops. The tomato (Solanum lycopersicum) is regarded as a model plant of the Solanaceae family. Recently, a number of tomato resources have been developed in parallel with the ongoing tomato genome sequencing project. In particular, a miniature cultivar, Micro-Tom, is regarded as a model system in tomato genomics, and a number of genomics resources in the Micro-Tom-background, such as ESTs and mutagenized lines, have been established by an international alliance. To accelerate the progress in tomato genomics, we developed a collection of fully-sequenced 13,227 Micro-Tom full-length cDNAs. By checking redundant sequences, coding sequences, and chimeric sequences, a set of 11,502 non-redundant full-length cDNAs (nrFLcDNAs) was generated. Analysis of untranslated regions demonstrated that tomato has longer 5'- and 3'-untranslated regions than most other plants but rice. Classification of functions of proteins predicted from the coding sequences demonstrated that nrFLcDNAs covered a broad range of functions. A comparison of nrFLcDNAs with genes of sixteen plants facilitated the identification of tomato genes that are not found in other plants, most of which did not have known protein domains. Mapping of the nrFLcDNAs onto currently available tomato genome sequences facilitated prediction of exon-intron structure. Introns of tomato genes were longer than those of Arabidopsis and rice. According to a comparison of exon sequences between the nrFLcDNAs and the tomato genome sequences, the frequency of nucleotide mismatch in exons between Micro-Tom and the genome-sequencing cultivar (Heinz 1706) was estimated to be 0.061%. The collection of Micro-Tom nrFLcDNAs generated in this study will serve as a valuable genomic tool for plant biologists to bridge the gap between basic and applied studies. The nrFLcDNA sequences will help annotation of the tomato whole-genome sequence and aid in tomato functional genomics and molecular breeding. Full-length cDNA sequences and their annotations are provided in the database KaFTom http://www.pgb.kazusa.or.jp/kaftom/ via the website of the National Bioresource Project Tomato http://tomato.nbrp.jp.

Characterization of full-length sequenced cDNA inserts (FLIcs) from Atlantic salmon (Salmo salar)

PubMed Central

Andreassen, Rune; Lunner, Sigbjørn; Høyheim, Bjørn

2009-01-01

Background Sequencing of the Atlantic salmon genome is now being planned by an international research consortium. Full-length sequenced inserts from cDNAs (FLIcs) are an important tool for correct annotation and clustering of the genomic sequence in any species. The large amount of highly similar duplicate sequences caused by the relatively recent genome duplication in the salmonid ancestor represents a particular challenge for the genome project. FLIcs will therefore be an extremely useful resource for the Atlantic salmon sequencing project. In addition to be helpful in order to distinguish between duplicate genome regions and in determining correct gene structures, FLIcs are an important resource for functional genomic studies and for investigation of regulatory elements controlling gene expression. In contrast to the large number of ESTs available, including the ESTs from 23 developmental and tissue specific cDNA libraries contributed by the Salmon Genome Project (SGP), the number of sequences where the full-length of the cDNA insert has been determined has been small. Results High quality full-length insert sequences from 560 pre-smolt white muscle tissue specific cDNAs were generated, accession numbers [GenBank: BT043497 - BT044056]. Five hundred and ten (91%) of the transcripts were annotated using Gene Ontology (GO) terms and 440 of the FLIcs are likely to contain a complete coding sequence (cCDS). The sequence information was used to identify putative paralogs, characterize salmon Kozak motifs, polyadenylation signal variation and to identify motifs likely to be involved in the regulation of particular genes. Finally, conserved 7-mers in the 3'UTRs were identified, of which some were identical to miRNA target sequences. Conclusion This paper describes the first Atlantic salmon FLIcs from a tissue and developmental stage specific cDNA library. We have demonstrated that many FLIcs contained a complete coding sequence (cCDS). This suggests that the remaining cDNA libraries generated by SGP represent a valuable cCDS FLIc source. The conservation of 7-mers in 3'UTRs indicates that these motifs are functionally important. Identity between some of these 7-mers and miRNA target sequences suggests that they are miRNA targets in Salmo salar transcripts as well. PMID:19878547

Intestinal Failure and Aberrant Lipid Metabolism in Patients With DGAT1 Deficiency.

PubMed

van Rijn, Jorik M; Chandra Ardy, Rico; Kuloğlu, Zarife; Härter, Bettina; van Haaften-Visser, Désirée Y; van der Doef, Hubert P J; van Hoesel, Marliek; Kansu, Aydan; van Vugt, Anke H M; Ng, Marini; Kokke, Freddy T M; Krolo, Ana; Başaran, Meryem Keçeli; Kaya, Neslihan Gurcan; Aksu, Aysel Ünlüsoy; Dalgıç, Buket; Ozcay, Figen; Baris, Zeren; Kain, Renate; Stigter, Edwin C A; Lichtenbelt, Klaske D; Massink, Maarten P G; Duran, Karen J; Verheij, Joke B G M; Lugtenberg, Dorien; Nikkels, Peter G J; Brouwer, Henricus G F; Verkade, Henkjan J; Scheenstra, Rene; Spee, Bart; Nieuwenhuis, Edward E S; Coffer, Paul J; Janecke, Andreas R; van Haaften, Gijs; Houwen, Roderick H J; Müller, Thomas; Middendorp, Sabine; Boztug, Kaan

2018-03-29

Congenital diarrheal disorders are rare inherited intestinal disorders characterized by intractable, sometimes life-threatening, diarrhea and nutrient malabsorption; some have been associated with mutations in diacylglycerol-acyltransferase 1 (DGAT1), which catalyzes formation of triacylglycerol from diacylglycerol and acyl-CoA. We investigated the mechanisms by which DGAT1 deficiency contributes to intestinal failure using patient-derived organoids. We collected blood samples from 10 patients, from 6 unrelated pedigrees, who presented with early-onset severe diarrhea and/or vomiting, hypoalbuminemia, and/or (fatal) protein-losing enteropathy with intestinal failure; we performed next-generation sequence analysis of DNA from 8 patients. Organoids were generated from duodenal biopsies from 3 patients and 3 healthy individuals (controls). Caco-2 cells and patient-derived dermal fibroblasts were transfected or transduced with vectors that express full-length or mutant forms of DGAT1 or full-length DGAT2. We performed CRISPR/Cas9-guided disruption of DGAT1 in control intestinal organoids. Cells and organoids were analyzed by immunoblot, immunofluorescence, flow cytometry, chromatography, quantitative real-time polymerase chain reaction, and for activities of caspases 3 and 7. In the 10 patients, we identified 5 bi-allelic loss-of-function mutations in DGAT1. In patient-derived fibroblasts and organoids, the mutations reduced expression of DGAT1 protein and altered triacylglycerol metabolism, resulting in decreased lipid droplet formation after oleic acid addition. Expression of full-length DGAT2 in patient-derived fibroblasts restored formation of lipid droplets. Organoids derived from patients with DGAT1 mutations were more susceptible to lipid-induced cell death than control organoids. We identified a large cohort of patients with congenital diarrheal disorders with mutations in DGAT1 that reduced expression of its product; dermal fibroblasts and intestinal organoids derived from these patients had altered lipid metabolism and were susceptible to lipid-induced cell death. Expression of full-length DGAT1 or DGAT2 restored normal lipid metabolism in these cells. These findings indicate the importance of DGAT1 in fat metabolism and lipotoxicity in the intestinal epithelium. A fat-free diet might serve as the first line of therapy for patients with reduced DGAT1 expression. It is important to identify genetic variants associated with congenital diarrheal disorders for proper diagnosis and selection of treatment strategies. Copyright © 2018 AGA Institute. Published by Elsevier Inc. All rights reserved.

Audiovisual Speech Web-Lab: an Internet teaching and research laboratory.

PubMed

Gordon, M S; Rosenblum, L D

2001-05-01

Internet resources now enable laboratories to make full-length experiments available on line. A handful of existing web sites offer users the ability to participate in experiments and generate usable data. We have integrated this technology into a web site that also provides full discussion of the theoretical and methodological aspects of the experiments using text and simple interactive demonstrations. The content of the web site (http://www.psych.ucr.edu/avspeech/lab) concerns audiovisual speech perception and its relation to face perception. The site is designed to be useful for users of multiple interests and levels of expertise.

A large-scale full-length cDNA analysis to explore the budding yeast transcriptome

PubMed Central

Miura, Fumihito; Kawaguchi, Noriko; Sese, Jun; Toyoda, Atsushi; Hattori, Masahira; Morishita, Shinichi; Ito, Takashi

2006-01-01

We performed a large-scale cDNA analysis to explore the transcriptome of the budding yeast Saccharomyces cerevisiae. We sequenced two cDNA libraries, one from the cells exponentially growing in a minimal medium and the other from meiotic cells. Both libraries were generated by using a vector-capping method that allows the accurate mapping of transcription start sites (TSSs). Consequently, we identified 11,575 TSSs associated with 3,638 annotated genomic features, including 3,599 ORFs, to suggest that most yeast genes have two or more TSSs. In addition, we identified 45 previously undescribed introns, including those affecting current ORF annotations and those spliced alternatively. Furthermore, the analysis revealed 667 transcription units in the intergenic regions and transcripts derived from antisense strands of 367 known features. We also found that 348 ORFs carry TSSs in their 3′-halves to generate sense transcripts starting from inside the ORFs. These results indicate that the budding yeast transcriptome is considerably more complex than previously thought, and it shares many recently revealed characteristics with the transcriptomes of mammals and other higher eukaryotes. Thus, the genome-wide active transcription that generates novel classes of transcripts appears to be an intrinsic feature of the eukaryotic cells. The budding yeast will serve as a versatile model for the studies on these aspects of transcriptome, and the full-length cDNA clones can function as an invaluable resource in such studies. PMID:17101987

Optimal design of vertebrate and insect sarcomeres.

PubMed

Otten, E

1987-01-01

This paper offers a model for the normalized length-tension relation of a muscle fiber based upon sarcomere design. Comparison with measurements published by Gordon et al. ('66) shows an accurate fit as long as the inhomogeneity of sarcomere length in a single muscle fiber is taken into account. Sequential change of filament length and the length of the cross-bridge-free zone leads the model to suggest that most vertebrate sarcomeres tested match the condition of optimal construction for the output of mechanical energy over a full sarcomere contraction movement. Joint optimization of all three morphometric parameters suggests that a slightly better (0.3%) design is theoretically possible. However, this theoretical sarcomere, optimally designed for the conversion of energy, has a low normalized contraction velocity; it provides a poorer match to the combined functional demands of high energy output and high contraction velocity than the real sarcomeres of vertebrates. The sarcomeres in fish myotomes appear to be built suboptimally for isometric contraction, but built optimally for that shortening velocity generating maximum power. During swimming, these muscles do indeed contract concentrically only. The sarcomeres of insect asynchronous flight muscles contract only slightly. They are not built optimally for maximum output of energy across the full range of contraction encountered in vertebrate sarcomeres, but are built almost optimally for the contraction range that they do in fact employ.

Effect of selection for growth rate on relative growth in rabbits.

PubMed

Pascual, M; Pla, M; Blasco, A

2008-12-01

The effect of selection for growth rate on relative growth of the rabbit body components was studied. Animals from the 18th generation of a line selected for growth rate were compared with a contemporary control group formed with offspring of embryos that were frozen at the seventh generation of selection of the same line. A total of 313 animals were slaughtered at 4, 9, 13, 20, and 40 wk old. The offal, organs, tissues, and retail cuts were weighed, and several carcass linear measurements were recorded. Huxley's allometric equations relating the weights of the components with respect to BW were fitted. Butterfield's quadratic equations relating the degree of maturity of the components and the degree of maturity of BW were also fitted. In most of the components studied, both models lead to similar patterns of growth. Blood was isometric or early maturing and skin was late maturing or isometric depending on the use of Huxley's or Butterfield's model. Full gastrointestinal tract, liver, kidneys, thoracic viscera, and head were early maturing, and the chilled carcass and reference carcass were late maturing. The retail cuts of the reference carcass showed isometry (forelegs) or late maturing growth (breast and ribs, loin, hind legs, and abdominal walls). Dissectible fat of the carcass and meat of the hind leg had a late development, whereas bone of the hind leg was early maturing. Lumbar circumference length was later maturing than the carcass length and thigh length. Sex did not affect the relative growth of most of the components. Butterfield's model showed that males had an earlier development of full gastrointestinal tract and later growth of kidneys than females. No effect of selection on the relative growth of any of the components studied was found, leading to similar patterns of growth and similar carcass composition at a given degree of maturity after 11 generations of selection for growth rate.

Comparison of Species Richness Estimates Obtained Using Nearly Complete Fragments and Simulated Pyrosequencing-Generated Fragments in 16S rRNA Gene-Based Environmental Surveys▿ †

PubMed Central

Youssef, Noha; Sheik, Cody S.; Krumholz, Lee R.; Najar, Fares Z.; Roe, Bruce A.; Elshahed, Mostafa S.

2009-01-01

Pyrosequencing-based 16S rRNA gene surveys are increasingly utilized to study highly diverse bacterial communities, with special emphasis on utilizing the large number of sequences obtained (tens to hundreds of thousands) for species richness estimation. However, it is not yet clear how the number of operational taxonomic units (OTUs) and, hence, species richness estimates determined using shorter fragments at different taxonomic cutoffs correlates with the number of OTUs assigned using longer, nearly complete 16S rRNA gene fragments. We constructed a 16S rRNA clone library from an undisturbed tallgrass prairie soil (1,132 clones) and used it to compare species richness estimates obtained using eight pyrosequencing candidate fragments (99 to 361 bp in length) and the nearly full-length fragment. Fragments encompassing the V1 and V2 (V1+V2) region and the V6 region (generated using primer pairs 8F-338R and 967F-1046R) overestimated species richness; fragments encompassing the V3, V7, and V7+V8 hypervariable regions (generated using primer pairs 338F-530R, 1046F-1220R, and 1046F-1392R) underestimated species richness; and fragments encompassing the V4, V5+V6, and V6+V7 regions (generated using primer pairs 530F-805R, 805F-1046R, and 967F-1220R) provided estimates comparable to those obtained with the nearly full-length fragment. These patterns were observed regardless of the alignment method utilized or the parameter used to gauge comparative levels of species richness (number of OTUs observed, slope of scatter plots of pairwise distance values for short and nearly complete fragments, and nonparametric and parametric species richness estimates). Similar results were obtained when analyzing three other datasets derived from soil, adult Zebrafish gut, and basaltic formations in the East Pacific Rise. Regression analysis indicated that these observed discrepancies in species richness estimates within various regions could readily be explained by the proportions of hypervariable, variable, and conserved base pairs within an examined fragment. PMID:19561178

An inducible amphipathic helix within the intrinsically disordered C terminus can participate in membrane curvature generation by peripherin-2/rds.

PubMed

Milstein, Michelle L; Kimler, Victoria A; Ghatak, Chiranjib; Ladokhin, Alexey S; Goldberg, Andrew F X

2017-05-12

Peripherin-2/rds is required for biogenesis of vertebrate photoreceptor outer segment organelles. Its localization at the high-curvature rim domains of outer segment disk membranes suggests that it may act to shape these structures; however, the molecular function of this protein is not yet resolved. Here, we apply biochemical, biophysical, and imaging techniques to elucidate the role(s) played by the protein's intrinsically disordered C-terminal domain and an incipient amphipathic α-helix contained within it. We investigated a deletion mutant lacking only this α-helix in stable cell lines and Xenopus laevis photoreceptors. We also studied a soluble form of the full-length ∼7-kDa cytoplasmic C terminus in cultured cells and purified from Escherichia coli The α-helical motif was not required for protein biosynthesis, tetrameric subunit assembly, tetramer polymerization, localization at disk rims, interaction with GARP2, or the generation of membrane curvature. Interestingly, however, loss of the helical motif up-regulated membrane curvature generation in cellulo , introducing the possibility that it may regulate this activity in photoreceptors. Furthermore, the incipient α-helix (within the purified soluble C terminus) partitioned into membranes only when its acidic residues were neutralized by protonation. This suggests that within the context of full-length peripherin-2/rds, partitioning would most likely occur at a bilayer interfacial region, potentially adjacent to the protein's transmembrane domains. In sum, this study significantly strengthens the evidence that peripherin-2/rds functions directly to shape the high-curvature rim domains of the outer segment disk and suggests that the protein's C terminus may modulate membrane curvature-generating activity present in other protein domains. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Understanding the Cellular Function of TRPV2 Channel through Generation of Specific Monoclonal Antibodies

PubMed Central

Cohen, Matthew R.; Huynh, Kevin W.; Cawley, Daniel; Moiseenkova-Bell, Vera Y.

2013-01-01

Transient receptor potential vanilloid 2 (TRPV2) is a Ca2+-permeable nonselective cation channel proposed to play a critical role in a wide array of cellular processes. Although TRPV2 surface expression was originally determined to be sensitive to growth factor signaling, regulated trafficking of TRPV2 has remained controversial. TRPV2 has proven difficult to study due to the lack of specific pharmacological tools to modulate channel activity; therefore, most studies of the cellular function of TRPV2 rely on immuno-detection techniques. Polyclonal antibodies against TRPV2 have not been properly validated and characterized, which may contribute to conflicting results regarding its function in the cell. Here, we developed monoclonal antibodies using full-length TRPV2 as an antigen. Extensive characterization of these antibodies and comparison to commonly used commercially available TRPV2 antibodies revealed that while monoclonal antibodies generated in our laboratory were suitable for detection of endogenous TRPV2 by western blot, immunoprecipitation and immunocytochemistry, the commercially available polyclonal antibodies we tested were not able to recognize endogenous TRPV2. We used our newly generated and validated TRPV2 antibodies to determine the effects of insulin-like growth factor 1 (IGF-1) on TRPV2 surface expression in heterologous and endogenous expression systems. We found that IGF-1 had little to no effect on trafficking and plasma membrane expression of TRPV2. Overall, these new TRPV2 monoclonal antibodies served to dispel the controversy of the effects of IGF-1 on TRPV2 plasma membrane expression and will clarify the role TRPV2 plays in cellular function. Furthermore, our strategy of using full-length tetrameric TRP channels may allow for the generation of antibodies against other TRP channels of unclear function. PMID:24392006

Understanding the cellular function of TRPV2 channel through generation of specific monoclonal antibodies.

PubMed

Cohen, Matthew R; Huynh, Kevin W; Cawley, Daniel; Moiseenkova-Bell, Vera Y

2013-01-01

Transient receptor potential vanilloid 2 (TRPV2) is a Ca(2+)-permeable nonselective cation channel proposed to play a critical role in a wide array of cellular processes. Although TRPV2 surface expression was originally determined to be sensitive to growth factor signaling, regulated trafficking of TRPV2 has remained controversial. TRPV2 has proven difficult to study due to the lack of specific pharmacological tools to modulate channel activity; therefore, most studies of the cellular function of TRPV2 rely on immuno-detection techniques. Polyclonal antibodies against TRPV2 have not been properly validated and characterized, which may contribute to conflicting results regarding its function in the cell. Here, we developed monoclonal antibodies using full-length TRPV2 as an antigen. Extensive characterization of these antibodies and comparison to commonly used commercially available TRPV2 antibodies revealed that while monoclonal antibodies generated in our laboratory were suitable for detection of endogenous TRPV2 by western blot, immunoprecipitation and immunocytochemistry, the commercially available polyclonal antibodies we tested were not able to recognize endogenous TRPV2. We used our newly generated and validated TRPV2 antibodies to determine the effects of insulin-like growth factor 1 (IGF-1) on TRPV2 surface expression in heterologous and endogenous expression systems. We found that IGF-1 had little to no effect on trafficking and plasma membrane expression of TRPV2. Overall, these new TRPV2 monoclonal antibodies served to dispel the controversy of the effects of IGF-1 on TRPV2 plasma membrane expression and will clarify the role TRPV2 plays in cellular function. Furthermore, our strategy of using full-length tetrameric TRP channels may allow for the generation of antibodies against other TRP channels of unclear function.

Walleye dermal sarcoma virus: expression of a full-length clone or the rv-cyclin (orf a) gene is cytopathic to the host and human tumor cells.

PubMed

Xu, Kun; Zhang, Ting Ting; Wang, Ling; Zhang, Cun Fang; Zhang, Long; Ma, Li Xia; Xin, Ying; Ren, Chong Hua; Zhang, Zhi Qiang; Yan, Qiang; Martineau, Daniel; Zhang, Zhi Ying

2013-02-01

Walleye dermal sarcoma virus (WDSV) is etiologically associated with a skin tumor, walleye dermal sarcoma (WDS), which develops in the fall and regresses in the spring. WDSV genome contains, in addition to gag, pol and env, three open reading frames (orfs) designated orf a (rv-cyclin), orf b and orf c. Unintegrated linear WDSV provirus DNA isolated from infected tumor cells was used to construct a full-length WDSV provirus clone pWDSV, while orf a was cloned into pSVK3 to construct the expression vector porfA. Stable co-transfection of a walleye cell line (W12) with pWDSV and pcDNA3 generated fewer and smaller G418-resistant colonies compared to the control. By Northern blot analysis, several small transcripts (2.8, 1.8, 1.2, and 0.8 kb) were detected using a WDSV LTR-specific probe. By RT-PCR and Southern blot analysis, three cDNAs (2.4, 1.6 and 0.8 kb) were identified, including both orf a and orf b messenger. Furthermore stable co-transfection of both a human lung adenocarcinoma cell line (SPC-A-1) and a cervical cancer cell line (HeLa) with pcDNA3 and ether porfA or pWDSV also generated fewer and smaller G418-resistant colonies. We conclude that expression of the full-length WDSV clone or the orf a gene inhibits the host fish and human tumor cell growth, and Orf A protein maybe a potential factor which contributes to the seasonal tumor development and regression. This is the first fish provirus clone that has been expressed in cell culture system, which will provide a new in vitro model for tumor research and oncotherapy study.

Extended core for motor/generator

DOEpatents

Shoykhet, Boris A.

2005-05-10

An extended stator core in a motor/generator can be utilized to mitigate losses in end regions of the core and a frame of the motor/generator. To mitigate the losses, the stator core can be extended to a length substantially equivalent to or greater than a length of a magnetically active portion in the rotor. Alternatively, a conventional length stator core can be utilized with a shortened magnetically active portion to mitigate losses in the motor/generator. To mitigate the losses in the core caused by stator winding, the core can be extended to a length substantially equivalent or greater than a length of stator winding.

Extended core for motor/generator

DOEpatents

Shoykhet, Boris A.

2006-08-22

An extended stator core in a motor/generator can be utilized to mitigate losses in end regions of the core and a frame of the motor/generator. To mitigate the losses, the stator core can be extended to a length substantially equivalent to or greater than a length of a magnetically active portion in the rotor. Alternatively, a conventional length stator core can be utilized with a shortened magnetically active portion to mitigate losses in the motor/generator. To mitigate the losses in the core caused by stator winding, the core can be extended to a length substantially equivalent or greater than a length of stator winding.

A Comparison of One Time Pad Random Key Generation using Linear Congruential Generator and Quadratic Congruential Generator

NASA Astrophysics Data System (ADS)

Apdilah, D.; Harahap, M. K.; Khairina, N.; Husein, A. M.; Harahap, M.

2018-04-01

One Time Pad algorithm always requires a pairing of the key for plaintext. If the length of keys less than a length of the plaintext, the key will be repeated until the length of the plaintext same with the length of the key. In this research, we use Linear Congruential Generator and Quadratic Congruential Generator for generating a random number. One Time Pad use a random number as a key for encryption and decryption process. Key will generate the first letter from the plaintext, we compare these two algorithms in terms of time speed encryption, and the result is a combination of OTP with LCG faster than the combination of OTP with QCG.

Unrelated sequences at the 5' end of mouse LINE-1 repeated elements define two distinct subfamilies.

PubMed Central

Wincker, P; Jubier-Maurin, V; Roizès, G

1987-01-01

Some full length members of the mouse long interspersed repeated DNA family L1Md have been shown to be associated at their 5' end with a variable number of tandem repetitions, the A repeats, that have been suggested to be transcription controlling elements. We report that the other type of repeat, named F, found at the 5' end of a few L1 elements is also an integral part of full length L1 copies. Sequencing shows that the F repeats are GC rich, and organized in tandem. The L1 copies associated with either A or F repeats can be correlated with two different subsets of L1 sequences distinguished by a series of variant nucleotides specific to each and by unassociated but frequent restriction sites. These findings suggest that sequence replacement has occurred at least once in 5' of L1Md, and is related to the generation of specific subfamilies. Images PMID:3684566

PRAPI: post-transcriptional regulation analysis pipeline for Iso-Seq.

PubMed

Gao, Yubang; Wang, Huiyuan; Zhang, Hangxiao; Wang, Yongsheng; Chen, Jinfeng; Gu, Lianfeng

2018-05-01

The single-molecule real-time (SMRT) isoform sequencing (Iso-Seq) based on Pacific Bioscience (PacBio) platform has received increasing attention for its ability to explore full-length isoforms. Thus, comprehensive tools for Iso-Seq bioinformatics analysis are extremely useful. Here, we present a one-stop solution for Iso-Seq analysis, called PRAPI to analyze alternative transcription initiation (ATI), alternative splicing (AS), alternative cleavage and polyadenylation (APA), natural antisense transcripts (NAT), and circular RNAs (circRNAs) comprehensively. PRAPI is capable of combining Iso-Seq full-length isoforms with short read data, such as RNA-Seq or polyadenylation site sequencing (PAS-seq) for differential expression analysis of NAT, AS, APA and circRNAs. Furthermore, PRAPI can annotate new genes and correct mis-annotated genes when gene annotation is available. Finally, PRAPI generates high-quality vector graphics to visualize and highlight the Iso-Seq results. The Dockerfile of PRAPI is available at http://www.bioinfor.org/tool/PRAPI. lfgu@fafu.edu.cn.

Characterization of a New HIV-1 CRF01_AE/ CRF07_BC recombinant virus in Tianjin, China.

PubMed

Zhou, Zhehua; Ma, Ping; Feng, Yi; Ou, Weidong; Qian, Jing; Gao, Liying; Zhang, Defa; Shao, Yiming; Wei, Min

2018-05-04

Human immunodeficiency virus (HIV) is notorious for its rapid evolving since its transmissions from money to human. Currently, HIV contains multiple subtypes, circulating recombinant forms (CRFs) and unique recombinant forms (URFs). Here, from an HIV-positive mother and her child in Tianjin, China, we identified a novel HIV-1 second-generation recombinant virus (TJ20170316 and TJ20170317) between CRF01_AE and CRF07_BC. Near full-length genomes were obtained from both samples, and they shared very close sequences, except some point mutations. Phylogenetic analyses of the near full-length genomes showed that they consist of CRF01_AE backbone and part CRF07_BC sequences. Recombinant Identification Program (RIP) and Simplot software identified four breakpoints in gag, pol, vif, tat genes in TJ20170316, totally different from other reported CRFs and URFs. The emergence of such URF in Tianjin, China, highlights the complexity of HIV-1 epidemic and more measures should be taken to prevent HIV transmissions.

Delta's Key to the Next Generation TOEFL[R] Test: Six Practice Tests for the iBT

ERIC Educational Resources Information Center

Gallagher, Nancy

2012-01-01

Six Practice Tests for the iBT gives students plenty of practice as they prepare for the Internet-based TOEFL (iBT) or the new form of the institutional TOEFL (ITP). This new book/audio set contains a concise description of the TOEFL and the types of questions in each section, as well as six full-length tests that have not been published before.…

Designing a Soluble Near Full-Length HIV-1 GP41 Trimer

DTIC Science & Technology

2012-11-26

envelope; gp41 trimer; bacteriophage T4 display; prehairpin fusion intermediate. Background: The envelope glycoprotein gp41 is a key component of...protein into trimers and defined oligomers. These gp41 trimers were displayed on bacteriophage T4 capsid nanoparticles by attaching to the small...Construction of the Expression Vectors —All the gp41 constructs were generated by splicing-by- overlap extension PCR using wild-type HXB2 gp41 DNA

MeSH indexing based on automatically generated summaries.

PubMed

Jimeno-Yepes, Antonio J; Plaza, Laura; Mork, James G; Aronson, Alan R; Díaz, Alberto

2013-06-26

MEDLINE citations are manually indexed at the U.S. National Library of Medicine (NLM) using as reference the Medical Subject Headings (MeSH) controlled vocabulary. For this task, the human indexers read the full text of the article. Due to the growth of MEDLINE, the NLM Indexing Initiative explores indexing methodologies that can support the task of the indexers. Medical Text Indexer (MTI) is a tool developed by the NLM Indexing Initiative to provide MeSH indexing recommendations to indexers. Currently, the input to MTI is MEDLINE citations, title and abstract only. Previous work has shown that using full text as input to MTI increases recall, but decreases precision sharply. We propose using summaries generated automatically from the full text for the input to MTI to use in the task of suggesting MeSH headings to indexers. Summaries distill the most salient information from the full text, which might increase the coverage of automatic indexing approaches based on MEDLINE. We hypothesize that if the results were good enough, manual indexers could possibly use automatic summaries instead of the full texts, along with the recommendations of MTI, to speed up the process while maintaining high quality of indexing results. We have generated summaries of different lengths using two different summarizers, and evaluated the MTI indexing on the summaries using different algorithms: MTI, individual MTI components, and machine learning. The results are compared to those of full text articles and MEDLINE citations. Our results show that automatically generated summaries achieve similar recall but higher precision compared to full text articles. Compared to MEDLINE citations, summaries achieve higher recall but lower precision. Our results show that automatic summaries produce better indexing than full text articles. Summaries produce similar recall to full text but much better precision, which seems to indicate that automatic summaries can efficiently capture the most important contents within the original articles. The combination of MEDLINE citations and automatically generated summaries could improve the recommendations suggested by MTI. On the other hand, indexing performance might be dependent on the MeSH heading being indexed. Summarization techniques could thus be considered as a feature selection algorithm that might have to be tuned individually for each MeSH heading.

Integrated optical phased arrays for quasi-Bessel-beam generation.

PubMed

Notaros, Jelena; Poulton, Christopher V; Byrd, Matthew J; Raval, Manan; Watts, Michael R

2017-09-01

Integrated optical phased arrays for generating quasi-Bessel beams are proposed and experimentally demonstrated in a CMOS-compatible platform. Owing to their elongated central beams, Bessel beams have applications in a range of fields, including multiparticle trapping and laser lithography. In this Letter, continuous Bessel theory is manipulated to formulate the phase and amplitude conditions necessary for generating free-space-propagating Bessel-Gauss beams using on-chip optical phased arrays. Discussion of the effects of select phased array parameters on the generated beam's figures of merit is included. A one-dimensional splitter-tree-based phased array architecture is modified to enable arbitrary passive control of the array's element phase and amplitude distributions. This architecture is used to experimentally demonstrate on-chip quasi-Bessel-beam generation with a ∼14  mm Bessel length and ∼30  μm power full width at half maximum.

DNA compaction by poly (amido amine) dendrimers of ammonia cored and ethylene diamine cored

NASA Astrophysics Data System (ADS)

Qamhieh, K.; Al-Shawwa, J.

2017-06-01

The complexes build-up of DNA and soft particles poly amidoamine (PAMAM) dendrimers of ammonia cored of generations (G1-G6) and ethylenediamine cored of generations (G1-G10) have been studied, using a new theoretical model developed by Qamhieh and coworkers. The model describes the interaction between linear polyelectrolyte (LPE) chain and ion-penetrable spheres. Many factors affecting LPE/dendrimer complex have been investigated such as dendrimer generation, the Bjerrum length, salt concentration, and rigidity of the LPE chain represented by the persistence length. It is found that the wrapping chain length around dendrimer increases by increasing dendrimer`s generation, Bjerrum length, and salt concentration, while decreases by increasing the persistence length of the LPE chain. Also we can conclude that the wrapping length of LPE chain around ethylenediamine cored dendrimers is larger than its length around ammonia cored dendrimers.

Tau Fibril Formation in Cultured Cells Compatible with a Mouse Model of Tauopathy.

PubMed

Matsumoto, Gen; Matsumoto, Kazuki; Kimura, Taeko; Suhara, Tetsuya; Higuchi, Makoto; Sahara, Naruhiko; Mori, Nozomu

2018-05-17

Neurofibrillary tangles composed of hyperphosphorylated tau protein are primarily neuropathological features of a number of neurodegenerative diseases collectively termed tauopathy. To understand the mechanisms underlying the cause of tauopathy, precise cellular and animal models are required. Recent data suggest that the transient introduction of exogenous tau can accelerate the development of tauopathy in the brains of non-transgenic and transgenic mice expressing wild-type human tau. However, the transmission mechanism leading to tauopathy is not fully understood. In this study, we developed cultured-cell models of tauopathy representing a human tauopathy. Neuro2a (N2a) cells containing propagative tau filaments were generated by introducing purified tau fibrils. These cell lines expressed full-length (2N4R) human tau and the green fluorescent protein (GFP)-fused repeat domain of tau with P301L mutation. Immunocytochemistry and super-resolution microscopic imaging revealed that tau inclusions exhibited filamentous morphology and were composed of both full-length and repeat domain fragment tau. Live-cell imaging analysis revealed that filamentous tau inclusions are transmitted to daughter cells, resulting in yeast-prion-like propagation. By a standard method of tau preparation, both full-length tau and repeat domain fragments were recovered in sarkosyl insoluble fraction. Hyperphosphorylation of full-length tau was confirmed by the immunoreactivity of phospho-Tau antibodies and mobility shifts by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). These properties were similar to the biochemical features of P301L mutated human tau in a mouse model of tauopathy. In addition, filamentous tau aggregates in cells barely co-localized with ubiquitins, suggesting that most tau aggregates were excluded from protein degradation systems, and thus propagated to daughter cells. The present cellular model of tauopathy will provide an advantage for dissecting the mechanisms of tau aggregation and degradation and be a powerful tool for drug screening to prevent tauopathy.

Expression of Kir7.1 and a Novel Kir7.1 Splice Variant in Native Human Retinal Pigment Epithelium

PubMed Central

Yang, Dongli; Swaminathan, Anuradha; Zhang, Xiaoming; Hughes, Bret A.

2009-01-01

Previous studies on bovine retinal pigment epithelium (RPE) established that Kir7.1 channels compose this epithelium’s large apical membrane K+ conductance. The purpose of this study was to determine whether Kir7.1 and potential Kir7.1 splice variants are expressed in native adult human RPE and, if so, to determine their function and how they are generated. RT-PCR analysis indicated that human RPE expresses full-length Kir7.1 and a novel Kir7.1 splice variant, designated Kir7.1S. Analysis of the human Kir7.1 gene (KCNJ13) organization revealed that it contains 3 exons, 2 introns, and a novel alternative 5′ splice site in exon 2. In human RPE, the alternative usage of two competing 5′ splice sites in exon 2 gives rise to transcripts encoding full-length Kir7.1 and Kir7.1S, which is predicted to encode a truncated protein. Real-time PCR indicated that Kir7.1 transcript is nearly as abundant as GAPDH mRNA in human RPE whereas Kir7.1S transcript expression is 4-fold lower. Western blot analysis showed that the splice variant is translated in Xenopus oocytes injected with Kir7.1S cRNA and revealed the expression of full-length Kir7.1 but not Kir7.1S in adult human RPE. Co-expression of Kir7.1 with Kir7.1S in Xenopus oocytes had no effect on either the kinetics or amplitude of Kir7.1 currents. This study confirms the expression of Kir7.1 in human RPE, identifies a Kir7.1 splice variant resulting in predicted changes in protein sequence, and indicates that there no functional interaction between this splice variant and full-length Kir7.1. PMID:18035352

Generation of Recombinant Ebola Viruses Using Reverse Genetics.

PubMed

Groseth, Allison

2017-01-01

Reverse genetics systems encompass a wide array of tools aimed at recapitulating some or all of the virus life cycle. In their most complete form, full-length clone systems allow us to use plasmid-encoded versions of the ribonucleoprotein (RNP) components to initiate the transcription and replication of a plasmid-encoded version of the complete viral genome, thereby initiating the complete virus life cycle and resulting in infectious virus. As such this approach is ideal for the generation of tailor-made recombinant filoviruses, which can be used to study virus biology. In addition, the generation of tagged and particularly fluorescent or luminescent viruses can be applied as tools for both diagnostic applications and for screening to identify novel countermeasures. Here we describe the generation and basic characterization of recombinant Ebola viruses rescued from cloned cDNA using a T7-driven system.

Systematic Analysis of Arabidopsis Organelles and a Protein Localization Database for Facilitating Fluorescent Tagging of Full-Length Arabidopsis Proteins1[W

PubMed Central

Li, Shijun; Ehrhardt, David W.; Rhee, Seung Y.

2006-01-01

Cells are organized into a complex network of subcellular compartments that are specialized for various biological functions. Subcellular location is an important attribute of protein function. To facilitate systematic elucidation of protein subcellular location, we analyzed experimentally verified protein localization data of 1,300 Arabidopsis (Arabidopsis thaliana) proteins. The 1,300 experimentally verified proteins are distributed among 40 different compartments, with most of the proteins localized to four compartments: mitochondria (36%), nucleus (28%), plastid (17%), and cytosol (13.3%). About 19% of the proteins are found in multiple compartments, in which a high proportion (36.4%) is localized to both cytosol and nucleus. Characterization of the overrepresented Gene Ontology molecular functions and biological processes suggests that the Golgi apparatus and peroxisome may play more diverse functions but are involved in more specialized processes than other compartments. To support systematic empirical determination of protein subcellular localization using a technology called fluorescent tagging of full-length proteins, we developed a database and Web application to provide preselected green fluorescent protein insertion position and primer sequences for all Arabidopsis proteins to study their subcellular localization and to store experimentally verified protein localization images, videos, and their annotations of proteins generated using the fluorescent tagging of full-length proteins technology. The database can be searched, browsed, and downloaded using a Web browser at http://aztec.stanford.edu/gfp/. The software can also be downloaded from the same Web site for local installation. PMID:16617091

Intersubunit distances in full-length, dimeric, bacterial phytochrome Agp1, as measured by pulsed electron-electron double resonance (PELDOR) between different spin label positions, remain unchanged upon photoconversion.

PubMed

Kacprzak, Sylwia; Njimona, Ibrahim; Renz, Anja; Feng, Juan; Reijerse, Edward; Lubitz, Wolfgang; Krauss, Norbert; Scheerer, Patrick; Nagano, Soshichiro; Lamparter, Tilman; Weber, Stefan

2017-05-05

Bacterial phytochromes are dimeric light-regulated histidine kinases that convert red light into signaling events. Light absorption by the N-terminal photosensory core module (PCM) causes the proteins to switch between two spectrally distinct forms, Pr and Pfr, thus resulting in a conformational change that modulates the C-terminal histidine kinase region. To provide further insights into structural details of photoactivation, we investigated the full-length Agp1 bacteriophytochrome from the soil bacterium Agrobacterium fabrum using a combined spectroscopic and modeling approach. We generated seven mutants suitable for spin labeling to enable application of pulsed EPR techniques. The distances between attached spin labels were measured using pulsed electron-electron double resonance spectroscopy to probe the arrangement of the subunits within the dimer. We found very good agreement of experimental and calculated distances for the histidine-kinase region when both subunits are in a parallel orientation. However, experimental distance distributions surprisingly showed only limited agreement with either parallel- or antiparallel-arranged dimer structures when spin labels were placed into the PCM region. This observation indicates that the arrangements of the PCM subunits in the full-length protein dimer in solution differ significantly from that in the PCM crystals. The pulsed electron-electron double resonance data presented here revealed either no or only minor changes of distance distributions upon Pr-to-Pfr photoconversion. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

What is the optimal way to prepare a Bell state using measurement and feedback?

NASA Astrophysics Data System (ADS)

Martin, Leigh; Sayrafi, Mahrud; Whaley, K. Birgitta

2017-12-01

Recent work has shown that the use of quantum feedback can significantly enhance both the speed and success rate of measurement-based remote entanglement generation, but it is generally unknown what feedback protocols are optimal for these tasks. Here we consider two common measurements that are capable of projecting into pairwise entangled states, namely half- and full-parity measurements of two qubits, and determine in each case a globally optimal protocol for generation of entanglement. For the half-parity measurement, we rederive a previously described protocol using more general methods and prove that it is globally optimal for several figures of merit, including maximal concurrence or fidelity and minimal time to reach a specified concurrence or fidelity. For the full-parity measurement, we derive a protocol for rapid entanglement generation related to that of (Hill, Ralph, Phys. Rev. A 77, 014305), and then map the dynamics of the concurrence of the state to the Bloch vector length of an effective qubit. This mapping allows us to prove several optimality results for feedback protocols with full-parity measurements. We further show that our full-parity protocol transfers entanglement optimally from one qubit to the other amongst all measurement-based schemes. The methods developed here will be useful for deriving feedback protocols and determining their optimality properties in many other quantum systems subject to measurement and unitary operations.

Terahertz generation by beating two Langmuir waves in a warm and collisional plasma

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Xiao-Bo; Qiao, Xin; Cheng, Li-Hong

2015-09-15

Terahertz (THz) radiation generated by beating of two Langmuir waves in a warm and collisional plasma is discussed theoretically. The critical angle between the two Langmuir waves and the critical wave-length (wave vector) of Langmuir waves for generating THz radiation are obtained analytically. Furthermore, the maximum radiation energy is obtained. We find that the critical angle, the critical wave-length, and the generated radiation energy strongly depend on plasma temperature and wave-length of the Langmuir waves. That is, the THz radiation generated by beating of two Langmuir waves in a warm and collisional plasma can be controlled by adjusting the plasmamore » temperature and the Langmuir wave-length.« less

A high HIV-1 strain variability in London, UK, revealed by full-genome analysis: Results from the ICONIC project.

PubMed

Yebra, Gonzalo; Frampton, Dan; Gallo Cassarino, Tiziano; Raffle, Jade; Hubb, Jonathan; Ferns, R Bridget; Waters, Laura; Tong, C Y William; Kozlakidis, Zisis; Hayward, Andrew; Kellam, Paul; Pillay, Deenan; Clark, Duncan; Nastouli, Eleni; Leigh Brown, Andrew J

2018-01-01

The ICONIC project has developed an automated high-throughput pipeline to generate HIV nearly full-length genomes (NFLG, i.e. from gag to nef) from next-generation sequencing (NGS) data. The pipeline was applied to 420 HIV samples collected at University College London Hospitals NHS Trust and Barts Health NHS Trust (London) and sequenced using an Illumina MiSeq at the Wellcome Trust Sanger Institute (Cambridge). Consensus genomes were generated and subtyped using COMET, and unique recombinants were studied with jpHMM and SimPlot. Maximum-likelihood phylogenetic trees were constructed using RAxML to identify transmission networks using the Cluster Picker. The pipeline generated sequences of at least 1Kb of length (median = 7.46Kb, IQR = 4.01Kb) for 375 out of the 420 samples (89%), with 174 (46.4%) being NFLG. A total of 365 sequences (169 of them NFLG) corresponded to unique subjects and were included in the down-stream analyses. The most frequent HIV subtypes were B (n = 149, 40.8%) and C (n = 77, 21.1%) and the circulating recombinant form CRF02_AG (n = 32, 8.8%). We found 14 different CRFs (n = 66, 18.1%) and multiple URFs (n = 32, 8.8%) that involved recombination between 12 different subtypes/CRFs. The most frequent URFs were B/CRF01_AE (4 cases) and A1/D, B/C, and B/CRF02_AG (3 cases each). Most URFs (19/26, 73%) lacked breakpoints in the PR+RT pol region, rendering them undetectable if only that was sequenced. Twelve (37.5%) of the URFs could have emerged within the UK, whereas the rest were probably imported from sub-Saharan Africa, South East Asia and South America. For 2 URFs we found highly similar pol sequences circulating in the UK. We detected 31 phylogenetic clusters using the full dataset: 25 pairs (mostly subtypes B and C), 4 triplets and 2 quadruplets. Some of these were not consistent across different genes due to inter- and intra-subtype recombination. Clusters involved 70 sequences, 19.2% of the dataset. The initial analysis of genome sequences detected substantial hidden variability in the London HIV epidemic. Analysing full genome sequences, as opposed to only PR+RT, identified previously undetected recombinants. It provided a more reliable description of CRFs (that would be otherwise misclassified) and transmission clusters.

Termination and read-through proteins encoded by genome segment 9 of Colorado tick fever virus.

PubMed

Mohd Jaafar, Fauziah; Attoui, Houssam; De Micco, Philippe; De Lamballerie, Xavier

2004-08-01

Genome segment 9 (Seg-9) of Colorado tick fever virus (CTFV) is 1884 bp long and contains a large open reading frame (ORF; 1845 nt in length overall), although a single in-frame stop codon (at nt 1052-1054) reduces the ORF coding capacity by approximately 40 %. However, analyses of highly conserved RNA sequences in the vicinity of the stop codon indicate that it belongs to a class of 'leaky terminators'. The third nucleotide positions in codons situated both before and after the stop codon, shows the highest variability, suggesting that both regions are translated during virus replication. This also suggests that the stop signal is functionally leaky, allowing read-through translation to occur. Indeed, both the truncated 'termination' protein and the full-length 'read-through' protein (VP9 and VP9', respectively) were detected in CTFV-infected cells, in cells transfected with a plasmid expressing only Seg-9 protein products, and in the in vitro translation products from undenatured Seg-9 ssRNA. The ratios of full-length and truncated proteins generated suggest that read-through may be down-regulated by other viral proteins. Western blot analysis of infected cells and purified CTFV showed that VP9 is a structural component of the virion, while VP9' is a non-structural protein.

Statistics of Radial Ship Extent as Seen by a Seeker

DTIC Science & Technology

2014-06-01

Auckland in pure and applied mathematics and physics, and a Master of Science in physics from the same university with a thesis in applied accelerator...does not demand contributions from two angle bins to one extent bin, unlike the rectangle; this is a very big advantage of the ellipse model. However...waveform that mimics the full length of a ship. This allows more economical use to be made of available false-target generation resources. I wish to

Commissioning a Megawatt-class Gyrotron with Collector Potential Depression

NASA Astrophysics Data System (ADS)

Lohr, J.; Cengher, M.; Gorelov, Y. A.; Ponce, D.; Prater, R.

2013-10-01

A 110 GHz depressed collector gyrotron has been installed on the DIII-D tokamak. The commissioning process rapidly achieved operation at full parameters, 45 A and 94 kV total voltage, with 29 kV depression. Although short pulse, 2 ms, factory testing demonstrated 1.2 MW at 41% electrical efficiency, long pulse testing at DIII-D achieved only 33% efficiency at full power parameters, for pulse lengths up to 10 s. Maximum generated power was ~950 kW, considerably below the 1.2 MW target. During attempts to increase the power at 5 s pulse length, it was noted that the collector cooling water was boiling. This led to the discovery that 14 of the 160 cooling channels in the collector had been blocked by braze material during manufacture of the tube. The locations of blocked channels were identified using infrared imaging of the outside of the collector during rapid changes in the cooling water temperature. Despite these difficulties, the rf beam itself was of very high quality and the stray rf found calorimetrically in the Matching Optics Unit, which couples the Gaussian rf beam to the waveguide, was only 2% of the generated power, about half that of our previous best quality high power beam. Details of the power measurements and collector observations will be presented. Work supported by the US DOE under DE-FC02-04ER54698.

HER2 isoforms co-expression differently tunes mammary tumor phenotypes affecting onset, vasculature and therapeutic response

PubMed Central

Balboni, Tania; Ianzano, Marianna L.; Laranga, Roberta; Landuzzi, Lorena; Giusti, Veronica; Ceccarelli, Claudio; Santini, Donatella; Taffurelli, Mario; Di Oto, Enrico; Asioli, Sofia; Amici, Augusto; Pupa, Serenella M.; De Giovanni, Carla; Tagliabue, Elda; Iezzi, Manuela; Nanni, Patrizia; Lollini, Pier-Luigi

2017-01-01

Full-length HER2 oncoprotein and splice variant Delta16 are co-expressed in human breast cancer. We studied their interaction in hybrid transgenic mice bearing human full-length HER2 and Delta16 (F1 HER2/Delta16) in comparison to parental HER2 and Delta16 transgenic mice. Mammary carcinomas onset was faster in F1 HER2/Delta16 and Delta16 than in HER2 mice, however tumor growth was slower, and metastatic spread was comparable in all transgenic mice. Full-length HER2 tumors contained few large vessels or vascular lacunae, whereas Delta16 tumors presented a more regular vascularization with numerous endothelium-lined small vessels. Delta16-expressing tumors showed a higher accumulation of i.v. injected doxorubicin than tumors expressing full-length HER2. F1 HER2/Delta16 tumors with high full-length HER2 expression made few large vessels, whereas tumors with low full-length HER2 and high Delta16 contained numerous small vessels and expressed higher levels of VEGF and VEGFR2. Trastuzumab strongly inhibited tumor onset in F1 HER2/Delta16 and Delta16 mice, but not in full-length HER2 mice. Addiction of F1 tumors to Delta16 was also shown by long-term stability of Delta16 levels during serial transplants, in contrast full-length HER2 levels underwent wide fluctuations. In conclusion, full-length HER2 leads to a faster tumor growth and to an irregular vascularization, whereas Delta16 leads to a faster tumor onset, with more regular vessels, which in turn could better transport cytotoxic drugs within the tumor, and to a higher sensitivity to targeted therapeutic agents. F1 HER2/Delta16 mice are a new immunocompetent mouse model, complementary to patient-derived xenografts, for studies of mammary carcinoma onset, prevention and therapy. PMID:28903354

Characterizing the turbulent porosity of stellar wind structure generated by the line-deshadowing instability

NASA Astrophysics Data System (ADS)

Owocki, Stanley P.; Sundqvist, Jon O.

2018-03-01

We analyse recent 2D simulations of the non-linear evolution of the line-deshadowing instability (LDI) in hot-star winds, to quantify how the associated highly clumped density structure can lead to a `turbulent porosity' reduction in continuum absorption and/or scattering. The basic method is to examine the statistical variations of mass column as a function of path length, and fit these to analytic forms that lead to simple statistical scalings for the associated mean extinction. A key result is that one can characterize porosity effects on continuum transport in terms of a single `turbulent porosity length', found here to scale as H ≈ (fcl - 1)a, where fcl ≡ 〈ρ2〉/〈ρ〉2 is the clumping factor in density ρ, and a is the density autocorrelation length. For continuum absorption or scattering in an optically thick layer, we find the associated effective reduction in opacity scales as ˜ 1/√{1+τ_H}, where τH ≡ κρH is the local optical thickness of this porosity length. For these LDI simulations, the inferred porosity lengths are small, only about a couple per cent of the stellar radius, H ≈ 0.02R*. For continuum processes like bound-free absorption of X-rays that are only marginally optically thick throughout the full stellar wind, this implies τH ≪ 1, and thus that LDI-generated porosity should have little effect on X-ray transport in such winds. The formalism developed here could however be important for understanding the porous regulation of continuum-driven, super-Eddington outflows from luminous blue variables.

Differences in the length of the carboxyl terminus mediate functional properties of neurokinin-1 receptor

PubMed Central

Lai, Jian-Ping; Lai, Saien; Tuluc, Florin; Tansky, Morris F.; Kilpatrick, Laurie E.; Leeman, Susan E.; Douglas, Steven D.

2008-01-01

The neurokinin-1 receptor (NK1R) has two naturally occurring forms that differ in the length of the carboxyl terminus: a full-length receptor consisting of 407 aa and a truncated receptor consisting of 311 aa. We examined whether there are differential signaling properties attributable to the carboxyl terminus of this receptor by using stably transfected human embryonic kidney (HEK293) cell lines that express either full-length or truncated NK1R. Substance P (SP) specifically triggered intracellular calcium increase in HEK293 cells expressing full-length NK1R but had no effect in the cells expressing the truncated NK1R. In addition, in cells expressing full-length NK1R, SP activated NF-κB and IL-8 mRNA expression, but in cells expressing the truncated NK1R, SP did not activate NF-κB, and it decreased IL-8 mRNA expression. In cells expressing full-length NK1R, SP stimulated phosphorylation of PKCδ but inhibited phosphorylation of PKCδ in cells expressing truncated NK1R. There are also differences in the timing of SP-induced ERK activation in cells expressing the two different forms of the receptor. Full-length NK1R activation of ERK was rapid (peak within 1–2 min), whereas truncated NK1R-mediated activation was slower (peak at 20–30 min). Thus, the carboxyl terminus of NK1R is the structural basis for differences in the functional properties of the full-length and truncated NK1R. These differences may provide important information toward the design of new NK1R receptor antagonists. PMID:18713853

Simulations and analysis of asteroid-generated tsunamis using the shallow water equations

NASA Astrophysics Data System (ADS)

Berger, M. J.; LeVeque, R. J.; Weiss, R.

2016-12-01

We discuss tsunami propagation for asteroid-generated air bursts and water impacts. We present simulations for a range of conditions using the GeoClaw simulation software. Examples include meteors that span 5 to 250 MT of kinetic energy, and use bathymetry from the U.S. coastline. We also study radially symmetric one-dimensional equations to better explore the nature and decay rate of waves generated by air burst pressure disturbances traveling at the speed of sound in air, which is much greater than the gravity wave speed of the tsunami generated. One-dimensional simulations along a transect of bathymetry are also used to explore the resolution needed for the full two-dimensional simulations, which are much more expensive even with the use of adaptive mesh refinement due to the short wave lengths of these tsunamis. For this same reason, shallow water equations may be inadequate and we also discuss dispersive effects.

Modeling of Ceiling Fire Spread and Thermal Radiation.

DTIC Science & Technology

1981-10-01

under a PMMA ceiling and flame lengths under an inert ceiling are found to be in reasonable agreement with full-scale behavior. Although fire spread...5 3 Flame Lengths under Full-Scale Ceilings 12 4 Correlation of Flame Length under Inert Ceilings 16 5 Correlation of Flame Length under No 234 Model...Ceilings 17 6 Correlation of Flame Length under No B8811 Model Ceilings 18 7 Correlation of Flame Length under No. 223 Model Ceilings 19 8

Impact of stent length on clinical outcomes of first-generation and new-generation drug-eluting stents.

PubMed

Konishi, Hirokazu; Miyauchi, Katsumi; Dohi, Tomotaka; Tsuboi, Shuta; Ogita, Manabu; Naito, Ryo; Kasai, Takatoshi; Tamura, Hiroshi; Okazaki, Shinya; Isoda, Kikuo; Daida, Hiroyuki

2016-04-01

The aim of this study is to compare first- and new-generation drug-eluting stents (DESs) which are implanted in long lesion. Stent length is known to be a predictor of adverse events after percutaneous coronary intervention (PCI), even with the first-generation DESs. The introduction of new-generation DESs has reduced the rates of adverse clinical events. However, the impact of stent length on long-term clinical outcomes is not well known. A total of 1181 consecutive patients who underwent PCI using either a first-generation DES (n = 885) or a new-generation DES (n = 296) between 2004 and 2011 were investigated. In each of the stent groups, the patients were divided into two groups by stent length (>32 and ≤32 mm) and compared. During the follow-up period, the incidence of major adverse cardiac events (MACEs) was significantly higher for patients with long stents implanted than with short stents (P < 0.01; log-rank test) in the first-generation DES group. However, there was no difference in the incidence of MACEs between the long- and short-stent groups in the new-generation DES group (P = 0.24; log-rank test). On multivariate Cox regression analysis, stent length was not associated with adverse events in the new-generation DES groups [hazard ratio (HR) 0.87; 95 % confidence interval (95 % CI) 0.71-1.04; P = 0.14]. Implanted stent length was significantly associated with a higher risk of MACEs in patients who received first-generation DESs, but not in patients who received the new-generation DESs.

Anti-sulfotyrosine antibodies

DOEpatents

Bertozzi, Carolyn R [Berkeley, CA; Kehoe, John [Saint Davids, PA; Bradbury, Andrew M [Santa Fe, NM

2009-09-15

The invention provides anti-sulfotyrosine specific antibodies capable of detecting and isolating polypeptides that are tyrosine-sulfated. The sulfotyrosine antibodies and antibody fragments of the invention may be used to discriminate between the non-sulfated and sulfated forms of such proteins, using any number of immunological assays, such ELISAs, immunoblots, Western Blots, immunoprecipitations, and the like. Using a phage-display system, single chain antibodies (scFvs) were generated and screened against tyrosine-sulfated synthetic peptide antigens, resulting in the isolation of scFvs that specifically recognize sulfotyrosine-containing peptides and/or demonstrate sulfotyrosine-specific binding in tyrosine sulfated proteins. The VH and VL genes from one such sulfotyrosine-specific scFv were employed to generate a full length, sulfotyrosine-specific immunoglobulin.

Polynucleotides encoding anti-sulfotyrosine antibodies

DOEpatents

Bertozzi, Carolyn R [Berkeley, CA; Kehoe, John [Saint Davids, PA; Bradbury, Andrew M [Santa Fe, NM

2011-01-11

The invention provides anti-sulfotyrosine specific antibodies capable of detecting and isolating polypeptides that are tyrosine-sulfated. The sulfotyrosine antibodies and antibody fragments of the invention may be used to discriminate between the non-sulfated and sulfated forms of such proteins, using any number of immunological assays, such ELISAs, immunoblots, Western Blots, immunoprecipitations, and the like. Using a phage-display system, single chain antibodies (scFvs) were generated and screened against tyrosine-sulfated synthetic peptide antigens, resulting in the isolation of scFvs that specifically recognize sulfotyrosine-containing peptides and/or demonstrate sulfotyrosine-specific binding in tyrosine sulfated proteins. The VH and VL genes from one such sulfotyrosine-specific scFv were employed to generate a full length, sulfotyrosine-specific immunoglobulin.

How muscle fiber lengths and velocities affect muscle force generation as humans walk and run at different speeds

PubMed Central

Arnold, Edith M.; Hamner, Samuel R.; Seth, Ajay; Millard, Matthew; Delp, Scott L.

2013-01-01

SUMMARY The lengths and velocities of muscle fibers have a dramatic effect on muscle force generation. It is unknown, however, whether the lengths and velocities of lower limb muscle fibers substantially affect the ability of muscles to generate force during walking and running. We examined this issue by developing simulations of muscle–tendon dynamics to calculate the lengths and velocities of muscle fibers from electromyographic recordings of 11 lower limb muscles and kinematic measurements of the hip, knee and ankle made as five subjects walked at speeds of 1.0–1.75 m s−1 and ran at speeds of 2.0–5.0 m s−1. We analyzed the simulated fiber lengths, fiber velocities and forces to evaluate the influence of force–length and force–velocity properties on force generation at different walking and running speeds. The simulations revealed that force generation ability (i.e. the force generated per unit of activation) of eight of the 11 muscles was significantly affected by walking or running speed. Soleus force generation ability decreased with increasing walking speed, but the transition from walking to running increased the force generation ability by reducing fiber velocities. Our results demonstrate the influence of soleus muscle architecture on the walk-to-run transition and the effects of muscle–tendon compliance on the plantarflexors' ability to generate ankle moment and power. The study presents data that permit lower limb muscles to be studied in unprecedented detail by relating muscle fiber dynamics and force generation to the mechanical demands of walking and running. PMID:23470656

VCSEL End-Pumped Passively Q-Switched Nd:YAG Laser with Adjustable Pulse Energy

DTIC Science & Technology

2011-02-28

entire VCSEL array. Neglecting lens aberrations, the focused spot diameter is given by focal length of the lens times the full divergence angle of the...pump intensity distribution generated by a pump-light-focusing lens . ©2011 Optical Society of America OCIS codes: (140.3530) Lasers Neodymium...Passive Q-Switch and Brewster Plate in a Pulsed Nd: YAG Laser,” IEEE J. Quantum Electron. 31(10), 1738–1741 (1995). 6. G. Xiao, and M. Bass, “A

On-Chip Sorting of Long Semiconducting Carbon Nanotubes for Multiple Transistors along an Identical Array.

PubMed

Otsuka, Keigo; Inoue, Taiki; Maeda, Etsuo; Kometani, Reo; Chiashi, Shohei; Maruyama, Shigeo

2017-11-28

Ballistic transport and sub-10 nm channel lengths have been achieved in transistors containing one single-walled carbon nanotube (SWNT). To fill the gap between single-tube transistors and high-performance logic circuits for the replacement of silicon, large-area, high-density, and purely semiconducting (s-) SWNT arrays are highly desired. Here we demonstrate the fabrication of multiple transistors along a purely semiconducting SWNT array via an on-chip purification method. Water- and polymer-assisted burning from site-controlled nanogaps is developed for the reliable full-length removal of metallic SWNTs with the damage to s-SWNTs minimized even in high-density arrays. All the transistors with various channel lengths show large on-state current and excellent switching behavior in the off-state. Since our method potentially provides pure s-SWNT arrays over a large area with negligible damage, numerous transistors with arbitrary dimensions could be fabricated using a conventional semiconductor process, leading to SWNT-based logic, high-speed communication, and other next-generation electronic devices.

Cultural Proficiency: Using Films to Get Groups Talking--and Listening--to One Another

ERIC Educational Resources Information Center

Nelson, Sarah W.; Guerra, Patricia L.

2009-01-01

Full-length films allow viewers to see the complexity and nuances of cultural interactions. Discussions following full-length films tend to be deeper and more insightful than those in response to a short clip. This makes watching full-length films an excellent strategy for helping teachers unpack beliefs, values, and stereotypes. In this article,…

Factors Influencing the Production of MFSV Full-Length Clone: Maize Fine Streak Virus Proteins in Drosophila S2 Cells

USDA-ARS?s Scientific Manuscript database

Maize fine streak virus (MFSV) is negative-sense RNA virus member of the genus Nucleorhabdovirus. Our goal is to determine whether Drosophila S2 cells can support the production of a full-length clone of MFSV. We have previously demonstrated that the full-length MFSV nucleoprotein (N) and phosphopro...

Decoding the massive genome of loblolly pine using haploid DNA and novel assembly strategies

PubMed Central

2014-01-01

Background The size and complexity of conifer genomes has, until now, prevented full genome sequencing and assembly. The large research community and economic importance of loblolly pine, Pinus taeda L., made it an early candidate for reference sequence determination. Results We develop a novel strategy to sequence the genome of loblolly pine that combines unique aspects of pine reproductive biology and genome assembly methodology. We use a whole genome shotgun approach relying primarily on next generation sequence generated from a single haploid seed megagametophyte from a loblolly pine tree, 20-1010, that has been used in industrial forest tree breeding. The resulting sequence and assembly was used to generate a draft genome spanning 23.2 Gbp and containing 20.1 Gbp with an N50 scaffold size of 66.9 kbp, making it a significant improvement over available conifer genomes. The long scaffold lengths allow the annotation of 50,172 gene models with intron lengths averaging over 2.7 kbp and sometimes exceeding 100 kbp in length. Analysis of orthologous gene sets identifies gene families that may be unique to conifers. We further characterize and expand the existing repeat library based on the de novo analysis of the repetitive content, estimated to encompass 82% of the genome. Conclusions In addition to its value as a resource for researchers and breeders, the loblolly pine genome sequence and assembly reported here demonstrates a novel approach to sequencing the large and complex genomes of this important group of plants that can now be widely applied. PMID:24647006

Deciphering of the Dual oxidase (Nox family) gene from kuruma shrimp, Marsupenaeus japonicus: full-length cDNA cloning and characterization.

PubMed

Inada, Mari; Kihara, Keisuke; Kono, Tomoya; Sudhakaran, Raja; Mekata, Tohru; Sakai, Masahiro; Yoshida, Terutoyo; Itami, Toshiaki

2013-02-01

In many physiological processes, including the innate immune system, free radicals such as nitric oxide (NO) and reactive oxygen species (ROS) play significant roles. In humans, 2 homologs of Dual oxidases (Duox) generate hydrogen peroxide (H(2)O(2)), which is a type of ROS. Here, we report the identification and characterization of a Duox from kuruma shrimp, Marsupenaeus japonicus. The full-length cDNA sequence of the M. japonicus Dual oxidase (MjDuox) gene contains 4695 bp and was generated using reverse transcriptase-polymerase chain reaction (RT-PCR) and random amplification of cDNA ends (RACE). The open reading frame of MjDuox encodes a protein of 1498 amino acids with an estimated mass of 173 kDa. In a homology analysis using amino acid sequences, MjDuox exhibited 69.3% sequence homology with the Duox of the red flour beetle, Tribolium castaneum. A transcriptional analysis revealed that the MjDuox mRNA is highly expressed in the gills of healthy kuruma shrimp. In the gills, MjDuox expression reached its peak 60 h after injection with WSSV and decreased to its normal level at 72 h. In gene knockdown experiments of free radical-generating enzymes, the survival rates decreased during the early stages of a white spot syndrome virus (WSSV) infection following the knockdown of the NADPH oxidase (MjNox) or MjDuox genes. In the present study, the identification, cloning and gene knockdown of the kuruma shrimp MjDuox are reported. Duoxes have been identified in vertebrates and some insects; however, few reports have investigated Duoxes in crustaceans. This study is the first to identify and clone a Dual oxidase from a crustacean species. Copyright © 2012 Elsevier Ltd. All rights reserved.

Fine Specificity of Plasmodium vivax Duffy Binding Protein Binding Engagement of the Duffy Antigen on Human Erythrocytes

PubMed Central

Siddiqui, Asim A.; Xainli, Jia; Schloegel, Jesse; Carias, Lenore; Ntumngia, Francis; Shoham, Menachem; Casey, Joanne L.; Foley, Michael; Adams, John H.

2012-01-01

Plasmodium vivax invasion of human erythrocytes requires interaction of the P. vivax Duffy binding protein (PvDBP) with its host receptor, the Duffy antigen (Fy) on the erythrocyte surface. Consequently, PvDBP is a leading vaccine candidate. The binding domain of PvDBP lies in a cysteine-rich portion of the molecule called region II (PvDBPII). PvDBPII contains three distinct subdomains based upon intramolecular disulfide bonding patterns. Subdomain 2 (SD2) is highly polymorphic and is thought to contain many key residues for binding to Fy, while SD1 and SD3 are comparatively conserved and their role in Fy binding is not well understood. To examine the relative contributions of the different subdomains to binding to Fy and their abilities to elicit strain-transcending binding-inhibitory antibodies, we evaluated recombinant proteins from SD1+2, SD2, SD3, and SD3+, which includes 24 residues of SD2. All of the recombinant subdomains, except for SD2, bound variably to human erythrocytes, with constructs containing SD3 showing the best binding. Antisera raised in laboratory animals against SD3, SD3+, and SD2+3 inhibited the binding of full-length PvDBPII, which is strain transcending, whereas antisera generated to SD1+2 and SD2 failed to generate blocking antibodies. All of the murine monoclonal antibodies generated to full-length PvDBPII that had significant binding-inhibitory activity recognized only SD3. Thus, SD3 binds Fy and elicits blocking antibodies, indicating that it contains residues critical to Fy binding that could be the basis of a strain-transcending candidate vaccine against P. vivax. PMID:22615246

Fine specificity of Plasmodium vivax Duffy binding protein binding engagement of the Duffy antigen on human erythrocytes.

PubMed

Siddiqui, Asim A; Xainli, Jia; Schloegel, Jesse; Carias, Lenore; Ntumngia, Francis; Shoham, Menachem; Casey, Joanne L; Foley, Michael; Adams, John H; King, Christopher L

2012-08-01

Plasmodium vivax invasion of human erythrocytes requires interaction of the P. vivax Duffy binding protein (PvDBP) with its host receptor, the Duffy antigen (Fy) on the erythrocyte surface. Consequently, PvDBP is a leading vaccine candidate. The binding domain of PvDBP lies in a cysteine-rich portion of the molecule called region II (PvDBPII). PvDBPII contains three distinct subdomains based upon intramolecular disulfide bonding patterns. Subdomain 2 (SD2) is highly polymorphic and is thought to contain many key residues for binding to Fy, while SD1 and SD3 are comparatively conserved and their role in Fy binding is not well understood. To examine the relative contributions of the different subdomains to binding to Fy and their abilities to elicit strain-transcending binding-inhibitory antibodies, we evaluated recombinant proteins from SD1+2, SD2, SD3, and SD3+, which includes 24 residues of SD2. All of the recombinant subdomains, except for SD2, bound variably to human erythrocytes, with constructs containing SD3 showing the best binding. Antisera raised in laboratory animals against SD3, SD3+, and SD2+3 inhibited the binding of full-length PvDBPII, which is strain transcending, whereas antisera generated to SD1+2 and SD2 failed to generate blocking antibodies. All of the murine monoclonal antibodies generated to full-length PvDBPII that had significant binding-inhibitory activity recognized only SD3. Thus, SD3 binds Fy and elicits blocking antibodies, indicating that it contains residues critical to Fy binding that could be the basis of a strain-transcending candidate vaccine against P. vivax.

Induction of humoral responses to BHV-1 glycoprotein D expressed by HSV-1 amplicon vectors

PubMed Central

Blanc, Andrea Maria; Berois, Mabel Beatriz; Tomé, Lorena Magalí; Epstein, Alberto L.

2012-01-01

Herpes simplex virus type-1 (HSV-1) amplicon vectors are versatile and useful tools for transferring genes into cells that are capable of stimulating a specific immune response to their expressed antigens. In this work, two HSV-1-derived amplicon vectors were generated. One of these expressed the full-length glycoprotein D (gD) of bovine herpesvirus 1 while the second expressed the truncated form of gD (gDtr) which lacked the trans-membrane region. After evaluating gD expression in the infected cells, the ability of both vectors to induce a specific gD immune response was tested in BALB/c mice that were intramuscularly immunized. Specific serum antibody responses were detected in mice inoculated with both vectors, and the response against truncated gD was higher than the response against full-length gD. These results reinforce previous findings that HSV-1 amplicon vectors can potentially deliver antigens to animals and highlight the prospective use of these vectors for treating infectious bovine rhinotracheitis disease. PMID:22437537

Detection and initial characterization of protein entities consisting of the HIV glycoprotein cytoplasmic C-terminal domain alone.

PubMed

Pfeiffer, Tanya; Ruppert, Thomas; Schaal, Heiner; Bosch, Valerie

2013-06-20

Employing antibodies against the cytoplasmic tail of the HIV-1 glycoprotein (Env-CT), in addition to gp160/gp41, we have identified several novel small Env proteins (<25kD) in HIV-1 transfected and infected cells. Mass spectrometric and mutational analyses show that two mechanisms contribute to their generation. Thus the protein, designated Tr-Env-CT (for truncated Env-CT), consists of the C-terminal 139 amino acids (aa) of Env (aa 718-856) with the N-terminal Q718 modified to pyroglutamic acid. It is likely derived from full-length Env protein by proteolytic processing. A further heterogeneous set of slightly larger proteins, termed Env-CT* species, are rather derived from spliced mRNAs containing only those Env C-terminal residues (aa 719-856) which overlap with the second tat and rev coding exons. They are N-terminally extended in the same reading frame. It is conceivable that essential Env-CT functions may be fulfilled by these novel species rather than by the full-length glycoprotein itself. Copyright © 2013 Elsevier Inc. All rights reserved.

Immunopurification of adenomatous polyposis coli (APC) proteins

PubMed Central

2013-01-01

Background The adenomatous polyposis coli (APC) tumour suppressor gene encodes a 2843 residue (310 kDa) protein. APC is a multifunctional protein involved in the regulation of β-catenin/Wnt signalling, cytoskeletal dynamics and cell adhesion. APC mutations occur in most colorectal cancers and typically result in truncation of the C-terminal half of the protein. Results In order to investigate the biophysical properties of APC, we have generated a set of monoclonal antibodies which enable purification of recombinant forms of APC. Here we describe the characterisation of these anti-APC monoclonal antibodies (APC-NT) that specifically recognise endogenous APC both in solution and in fixed cells. Full-length APC(1–2843) and cancer-associated, truncated APC proteins, APC(1–1638) and APC(1–1311) were produced in Sf9 insect cells. Conclusions Recombinant APC proteins were purified using a two-step affinity approach using our APC-NT antibodies. The purification of APC proteins provides the basis for detailed structure/function analyses of full-length, cancer-truncated and endogenous forms of the protein. PMID:24156781

Lactobacillus bulgaricus Proteinase Expressed in Lactococcus lactis Is a Powerful Carrier for Cell Wall-Associated and Secreted Bovine β-Lactoglobulin Fusion Proteins

PubMed Central

Bernasconi, Eric; Germond, Jacques-Edouard; Delley, Michèle; Fritsché, Rodolphe; Corthésy, Blaise

2002-01-01

Lactic acid bacteria have a good potential as agents for the delivery of heterologous proteins to the gastrointestinal mucosa and thus for the reequilibration of inappropriate immune responses to food antigens. Bovine β-lactoglobulin (BLG) is considered a major allergen in cow's milk allergy. We have designed recombinant Lactococcus lactis expressing either full-length BLG or BLG-derived octapeptide T6 (IDALNENK) as fusions with Lactobacillus bulgaricus extracellular proteinase (PrtB). In addition to constructs encoding full-length PrtB for the targeting of heterologous proteins to the cell surface, we generated vectors aiming at the release into the medium of truncated PrtB derivatives lacking 100 (PrtB∂, PrtB∂-BLG, and PrtB∂-T6) or 807 (PrtBΔ) C-terminal amino acids. Expression of recombinant products was confirmed using either anti-PrtB, anti-BLG, or anti-peptide T6 antiserum. All forms of the full-length and truncated recombinant products were efficiently translocated, irrespective of the presence of eucaryotic BLG sequences in the fusion proteins. L. lactis expressing PrtB∂-BLG yielded up to 170 μg per 109 CFU in the culture supernatant and 9 μg per 109 CFU at the bacterial cell surface within 14 h. Therefore, protein fusions relying on the use of PrtB gene products are adequate for concomitant cell surface display and secretion by recombinant L. lactis and thus may ensure maximal bioavailability of the eucaryotic antigen in the gut-associated lymphoid tissue. PMID:12039750

Internally deleted WNV genomes isolated from exotic birds in New Mexico: function in cells, mosquitoes, and mice.

PubMed

Pesko, Kendra N; Fitzpatrick, Kelly A; Ryan, Elizabeth M; Shi, Pei-Yong; Zhang, Bo; Lennon, Niall J; Newman, Ruchi M; Henn, Matthew R; Ebel, Gregory D

2012-05-25

Most RNA viruses exist in their hosts as a heterogeneous population of related variants. Due to error prone replication, mutants are constantly generated which may differ in individual fitness from the population as a whole. Here we characterize three WNV isolates that contain, along with full-length genomes, mutants with large internal deletions to structural and nonstructural protein-coding regions. The isolates were all obtained from lorikeets that died from WNV at the Rio Grande Zoo in Albuquerque, NM between 2005 and 2007. The deletions are approximately 2kb, in frame, and result in the elimination of the complete envelope, and portions of the prM and NS-1 proteins. In Vero cell culture, these internally deleted WNV genomes function as defective interfering particles, reducing the production of full-length virus when introduced at high multiplicities of infection. In mosquitoes, the shortened WNV genomes reduced infection and dissemination rates, and virus titers overall, and were not detected in legs or salivary secretions at 14 or 21 days post-infection. In mice, inoculation with internally deleted genomes did not attenuate pathogenesis relative to full-length or infectious clone derived virus, and shortened genomes were not detected in mice at the time of death. These observations provide evidence that large deletions may occur within flavivirus populations more frequently than has generally been appreciated and suggest that they impact population phenotype minimally. Additionally, our findings suggest that highly similar mutants may frequently occur in particular vertebrate hosts. Copyright © 2012 Elsevier Inc. All rights reserved.

RiceFOX: a database of Arabidopsis mutant lines overexpressing rice full-length cDNA that contains a wide range of trait information to facilitate analysis of gene function.

PubMed

Sakurai, Tetsuya; Kondou, Youichi; Akiyama, Kenji; Kurotani, Atsushi; Higuchi, Mieko; Ichikawa, Takanari; Kuroda, Hirofumi; Kusano, Miyako; Mori, Masaki; Saitou, Tsutomu; Sakakibara, Hitoshi; Sugano, Shoji; Suzuki, Makoto; Takahashi, Hideki; Takahashi, Shinya; Takatsuji, Hiroshi; Yokotani, Naoki; Yoshizumi, Takeshi; Saito, Kazuki; Shinozaki, Kazuo; Oda, Kenji; Hirochika, Hirohiko; Matsui, Minami

2011-02-01

Identification of gene function is important not only for basic research but also for applied science, especially with regard to improvements in crop production. For rapid and efficient elucidation of useful traits, we developed a system named FOX hunting (Full-length cDNA Over-eXpressor gene hunting) using full-length cDNAs (fl-cDNAs). A heterologous expression approach provides a solution for the high-throughput characterization of gene functions in agricultural plant species. Since fl-cDNAs contain all the information of functional mRNAs and proteins, we introduced rice fl-cDNAs into Arabidopsis plants for systematic gain-of-function mutation. We generated >30,000 independent Arabidopsis transgenic lines expressing rice fl-cDNAs (rice FOX Arabidopsis mutant lines). These rice FOX Arabidopsis lines were screened systematically for various criteria such as morphology, photosynthesis, UV resistance, element composition, plant hormone profile, metabolite profile/fingerprinting, bacterial resistance, and heat and salt tolerance. The information obtained from these screenings was compiled into a database named 'RiceFOX'. This database contains around 18,000 records of rice FOX Arabidopsis lines and allows users to search against all the observed results, ranging from morphological to invisible traits. The number of searchable items is approximately 100; moreover, the rice FOX Arabidopsis lines can be searched by rice and Arabidopsis gene/protein identifiers, sequence similarity to the introduced rice fl-cDNA and traits. The RiceFOX database is available at http://ricefox.psc.riken.jp/.

RiceFOX: A Database of Arabidopsis Mutant Lines Overexpressing Rice Full-Length cDNA that Contains a Wide Range of Trait Information to Facilitate Analysis of Gene Function

PubMed Central

Sakurai, Tetsuya; Kondou, Youichi; Akiyama, Kenji; Kurotani, Atsushi; Higuchi, Mieko; Ichikawa, Takanari; Kuroda, Hirofumi; Kusano, Miyako; Mori, Masaki; Saitou, Tsutomu; Sakakibara, Hitoshi; Sugano, Shoji; Suzuki, Makoto; Takahashi, Hideki; Takahashi, Shinya; Takatsuji, Hiroshi; Yokotani, Naoki; Yoshizumi, Takeshi; Saito, Kazuki; Shinozaki, Kazuo; Oda, Kenji; Hirochika, Hirohiko; Matsui, Minami

2011-01-01

Identification of gene function is important not only for basic research but also for applied science, especially with regard to improvements in crop production. For rapid and efficient elucidation of useful traits, we developed a system named FOX hunting (Full-length cDNA Over-eXpressor gene hunting) using full-length cDNAs (fl-cDNAs). A heterologous expression approach provides a solution for the high-throughput characterization of gene functions in agricultural plant species. Since fl-cDNAs contain all the information of functional mRNAs and proteins, we introduced rice fl-cDNAs into Arabidopsis plants for systematic gain-of-function mutation. We generated >30,000 independent Arabidopsis transgenic lines expressing rice fl-cDNAs (rice FOX Arabidopsis mutant lines). These rice FOX Arabidopsis lines were screened systematically for various criteria such as morphology, photosynthesis, UV resistance, element composition, plant hormone profile, metabolite profile/fingerprinting, bacterial resistance, and heat and salt tolerance. The information obtained from these screenings was compiled into a database named ‘RiceFOX’. This database contains around 18,000 records of rice FOX Arabidopsis lines and allows users to search against all the observed results, ranging from morphological to invisible traits. The number of searchable items is approximately 100; moreover, the rice FOX Arabidopsis lines can be searched by rice and Arabidopsis gene/protein identifiers, sequence similarity to the introduced rice fl-cDNA and traits. The RiceFOX database is available at http://ricefox.psc.riken.jp/. PMID:21186176

MeSH indexing based on automatically generated summaries

PubMed Central

2013-01-01

Background MEDLINE citations are manually indexed at the U.S. National Library of Medicine (NLM) using as reference the Medical Subject Headings (MeSH) controlled vocabulary. For this task, the human indexers read the full text of the article. Due to the growth of MEDLINE, the NLM Indexing Initiative explores indexing methodologies that can support the task of the indexers. Medical Text Indexer (MTI) is a tool developed by the NLM Indexing Initiative to provide MeSH indexing recommendations to indexers. Currently, the input to MTI is MEDLINE citations, title and abstract only. Previous work has shown that using full text as input to MTI increases recall, but decreases precision sharply. We propose using summaries generated automatically from the full text for the input to MTI to use in the task of suggesting MeSH headings to indexers. Summaries distill the most salient information from the full text, which might increase the coverage of automatic indexing approaches based on MEDLINE. We hypothesize that if the results were good enough, manual indexers could possibly use automatic summaries instead of the full texts, along with the recommendations of MTI, to speed up the process while maintaining high quality of indexing results. Results We have generated summaries of different lengths using two different summarizers, and evaluated the MTI indexing on the summaries using different algorithms: MTI, individual MTI components, and machine learning. The results are compared to those of full text articles and MEDLINE citations. Our results show that automatically generated summaries achieve similar recall but higher precision compared to full text articles. Compared to MEDLINE citations, summaries achieve higher recall but lower precision. Conclusions Our results show that automatic summaries produce better indexing than full text articles. Summaries produce similar recall to full text but much better precision, which seems to indicate that automatic summaries can efficiently capture the most important contents within the original articles. The combination of MEDLINE citations and automatically generated summaries could improve the recommendations suggested by MTI. On the other hand, indexing performance might be dependent on the MeSH heading being indexed. Summarization techniques could thus be considered as a feature selection algorithm that might have to be tuned individually for each MeSH heading. PMID:23802936

The role of plasma density scale length on the laser pulse propagation and scattering in relativistic regime

NASA Astrophysics Data System (ADS)

Pishdast, Masoud; Ghasemi, Seyed Abolfazl; Yazdanpanah, Jamal Aldin

2017-10-01

The role of plasma density scale length on two short and long laser pulse propagation and scattering in under dense plasma have been investigated in relativistic regime using 1 D PIC simulation. In our simulation, different density scale lengths and also two short and long pulse lengths with temporal pulse duration τL = 60 fs and τL = 300 fs , respectively have been used. It is found that laser pulse length and density scale length have considerable effects on the energetic electron generation. The analysis of total radiation spectrum reveals that, for short laser pulses and with reducing density scale length, more unstable electromagnetic modes grow and strong longitudinal electric field generates which leads to the generation of more energetic plasma particles. Meanwhile, the dominant scattering mechanism is Raman scattering and tends to Thomson scattering for longer laser pulse.

Optimization for Single-Spike X-Ray FELs at LCLS with a Low Charge Beam

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, L.; Ding, Y.; Huang, Z.

2011-12-14

The Linac Coherent Light Source is an x-ray free-electron laser at the SLAC National Accelerator Laboratory, which is operating at x-ray wavelengths of 20-1.2 Angstrom with peak brightness nearly ten orders of magnitude beyond conventional synchrotron radiation sources. At the low charge operation mode (20 pC), the x-ray pulse length can be <10 fs. In this paper we report our numerical optimization and simulations to produce even shorter x-ray pulses by optimizing the machine and undulator setup at 20 pC charge. In the soft x-ray regime, with combination of slotted-foil or undulator taper, a single spike x-ray pulse is achievablemore » with peak FEL power of a few 10s GW. Linac Coherent Light Source (LCLS), the world's first hard x-ray Free electron laser (FEL), has started operation since 2009. With nominal operation charge of 250 pC, the generated x-ray pulse length is from 70 fs to a few hundred fs. This marks the beginning of a new era of ultrashort x-ray sciences. In addition, a low charge (20pC) operation mode has also been established. Since the collective effects are reduced at the low charge mode, we can increase the compression factor and still achieve a few kA peak current. The expected electron beam and x-ray pulses are less than 10 fs. There are growing interests in even shorter x-ray pulses, such as fs to sub-fs regime. One of the simple solutions is going to even lower charge. As discussed, single-spike x-ray pulses can be generated using 1 pC charge. However, this charge level is out of the present LCLS diagnostic range. 20 pC is a reasonable operation charge at LCLS, based on the present diagnostic system. At 20 pC in the soft x-ray wavelength regime, we have experimentally demonstrated that FEL can work at undercompression or over-compression mode, such as 1 degree off the full-compression; at full-compression, however, there is almost no lasing. In hard x-ray wavelength regime, we observed that there are reasonable photons generated even at full-compression mode, although the photon number is less than that from under-compression or over-compression mode. Since we cannot measure the x-ray pulse length at this time scale, the machine is typically optimized for generating maximum photons, not minimum pulse length. In this paper, we study the methods of producing femtosecond (or single-spike) x-ray pulses at LCLS with 20 pC charge, based on start-to-end simulations. Figure 1 shows a layout of LCLS. The compression in the second bunch compressor (BC2) determines the final e-beam bunch length. However, the laser heater, dog-leg after the main linac (DL2) and collective effects also affect the final bunch length. To adjust BC2 compression, we can either change the L2 phase or BC2 R{sub 56}. In this paper we only tune L2 phase while keep BC2 R{sub 56} fixed. For the start-to-end simulations, we used IMPACT-T and ELEGANT tracking from the photocathode to the entrance of the undulator, after that the FEL radiation was simulated with GENESIS. IMPACT-T tracks about 10{sup 6} particles in the injector part until 135 MeV, including 3D space charge force. The output particles from IMPACT-T are smoothed and increased to 12 x 10{sup 6} to reduce high-frequency numerical noise for subsequent ELEGANT simulations, which include linear and nonlinear transport effects, a 1D transient model of CSR, and longitudinal space charge effects, as well as geometric and resistive wake fields in the accelerator. In GENESIS part, the longitudinal wake field from undulator chamber and longitudinal space field are also included.« less

Generation of Infectious Poliovirus with Altered Genetic Information from Cloned cDNA.

PubMed

Bujaki, Erika

2016-01-01

The effect of specific genetic alterations on virus biology and phenotype can be studied by a great number of available assays. The following method describes the basic protocol to generate infectious poliovirus with altered genetic information from cloned cDNA in cultured cells.The example explained here involves generation of a recombinant poliovirus genome by simply replacing a portion of the 5' noncoding region with a synthetic gene by restriction cloning. The vector containing the full length poliovirus genome and the insert DNA with the known mutation(s) are cleaved for directional cloning, then ligated and transformed into competent bacteria. The recombinant plasmid DNA is then propagated in bacteria and transcribed to RNA in vitro before RNA transfection of cultured cells is performed. Finally, viral particles are recovered from the cell culture.

Changes in passive tension of muscle in humans and animals after eccentric exercise

PubMed Central

Whitehead, N P; Weerakkody, N S; Gregory, J E; Morgan, D L; Proske, U

2001-01-01

This is a report of experiments on ankle extensor muscles of human subjects and a parallel series on the medial gastrocnemius of the anaesthetised cat, investigating the origin of the rise in passive tension after a period of eccentric exercise. Subjects exercised their triceps surae of one leg eccentrically by walking backwards on an inclined, forward-moving treadmill. Concentric exercise required walking forwards on a backwards-moving treadmill. For all subjects the other leg acted as a control. Immediately after both eccentric and concentric exercise there was a significant drop in peak active torque, but only after eccentric exercise was this accompanied by a shift in optimum angle for torque generation and a rise in passive torque. In the eccentrically exercised group some swelling and soreness developed but not until 24 h post-exercise. In the animal experiments the contracting muscle was stretched by 6 mm at 50 mm s−1 over a length range symmetrical about the optimum length for tension generation. Measurements of passive tension were made before and after the eccentric contractions, using small stretches to a range of muscle lengths, or with large stretches covering the full physiological range. After 150 eccentric contractions, passive tension was significantly elevated over most of the range of lengths. Measurements of work absorption during stretch-release cycles showed significant increases after the contractions. It is suggested that the rise in passive tension in both human and animal muscles after eccentric contractions is the result of development of injury contractures in damaged muscle fibres. PMID:11389215

Feasibility of Using Neural Network Models to Accelerate the Testing of Mechanical Systems

NASA Technical Reports Server (NTRS)

Fusaro, Robert L.

1998-01-01

Verification testing is an important aspect of the design process for mechanical mechanisms, and full-scale, full-length life testing is typically used to qualify any new component for use in space. However, as the required life specification is increased, full-length life tests become more costly and lengthen the development time. At the NASA Lewis Research Center, we theorized that neural network systems may be able to model the operation of a mechanical device. If so, the resulting neural network models could simulate long-term mechanical testing with data from a short-term test. This combination of computer modeling and short-term mechanical testing could then be used to verify the reliability of mechanical systems, thereby eliminating the costs associated with long-term testing. Neural network models could also enable designers to predict the performance of mechanisms at the conceptual design stage by entering the critical parameters as input and running the model to predict performance. The purpose of this study was to assess the potential of using neural networks to predict the performance and life of mechanical systems. To do this, we generated a neural network system to model wear obtained from three accelerated testing devices: 1) A pin-on-disk tribometer; 2) A line-contact rub-shoe tribometer; 3) A four-ball tribometer.

Genotyping of the fish rhabdovirus, viral haemorrhagic septicaemia virus, by restriction fragment length polymorphisms

USGS Publications Warehouse

Einer-Jensen, Katja; Winton, James R.; Lorenzen, Niels

2005-01-01

The aim of this study was to develop a standardized molecular assay that used limited resources and equipment for routine genotyping of isolates of the fish rhabdovirus, viral haemorrhagic septicaemia virus (VHSV). Computer generated restriction maps, based on 62 unique full-length (1524 nt) sequences of the VHSV glycoprotein (G) gene, were used to predict restriction fragment length polymorphism (RFLP) patterns that were subsequently grouped and compared with a phylogenetic analysis of the G-gene sequences of the same set of isolates. Digestion of PCR amplicons from the full-lengthG-gene by a set of three restriction enzymes was predicted to accurately enable the assignment of the VHSV isolates into the four major genotypes discovered to date. Further sub-typing of the isolates into the recently described sub-lineages of genotype I was possible by applying three additional enzymes. Experimental evaluation of the method consisted of three steps: (i) RT-PCR amplification of the G-gene of VHSV isolates using purified viral RNA as template, (ii) digestion of the PCR products with a panel of restriction endonucleases and (iii) interpretation of the resulting RFLP profiles. The RFLP analysis was shown to approximate the level of genetic discrimination obtained by other, more labour-intensive, molecular techniques such as the ribonuclease protection assay or sequence analysis. In addition, 37 previously uncharacterised isolates from diverse sources were assigned to specific genotypes. While the assay was able to distinguish between marine and continental isolates of VHSV, the differences did not correlate with the pathogenicity of the isolates.

Subtraction of cap-trapped full-length cDNA libraries to select rare transcripts.

PubMed

Hirozane-Kishikawa, Tomoko; Shiraki, Toshiyuki; Waki, Kazunori; Nakamura, Mari; Arakawa, Takahiro; Kawai, Jun; Fagiolini, Michela; Hensch, Takao K; Hayashizaki, Yoshihide; Carninci, Piero

2003-09-01

The normalization and subtraction of highly expressed cDNAs from relatively large tissues before cloning dramatically enhanced the gene discovery by sequencing for the mouse full-length cDNA encyclopedia, but these methods have not been suitable for limited RNA materials. To normalize and subtract full-length cDNA libraries derived from limited quantities of total RNA, here we report a method to subtract plasmid libraries excised from size-unbiased amplified lambda phage cDNA libraries that avoids heavily biasing steps such as PCR and plasmid library amplification. The proportion of full-length cDNAs and the gene discovery rate are high, and library diversity can be validated by in silico randomization.

[Cloning and sequence analysis of full-length cDNA of secoisolariciresinol dehydrogenase of Dysosma versipellis].

PubMed

Xu, Li; Ding, Zhi-Shan; Zhou, Yun-Kai; Tao, Xue-Fen

2009-06-01

To obtain the full-length cDNA sequence of Secoisolariciresinol Dehydrogenase gene from Dysosma versipellis by RACE PCR,then investigate the character of Secoisolariciresinol Dehydrogenase gene. The full-length cDNA sequence of Secoisolariciresinol Dehydrogenase gene was obtained by 3'-RACE and 5'-RACE from Dysosma versipellis. We first reported the full cDNA sequences of Secoisolariciresinol Dehydrogenase in Dysosma versipellis. The acquired gene was 991bp in full length, including 5' untranslated region of 42bp, 3' untranslated region of 112bp with Poly (A). The open reading frame (ORF) encoding 278 amino acid with molecular weight 29253.3 Daltons and isolectric point 6.328. The gene accession nucleotide sequence number in GeneBank was EU573789. Semi-quantitative RT-PCR analysis revealed that the Secoisolariciresinol Dehydrogenase gene was highly expressed in stem. Alignment of the amino acid sequence of Secoisolariciresinol Dehydrogenase indicated there may be some significant amino acid sequence difference among different species. Obtain the full-length cDNA sequence of Secoisolariciresinol Dehydrogenase gene from Dysosma versipellis.

Use of Dried Blood Spots to Elucidate Full-Length Transmitted/Founder HIV-1 Genomes

PubMed Central

Salazar-Gonzalez, Jesus F.; Salazar, Maria G.; Tully, Damien C.; Ogilvie, Colin B.; Learn, Gerald H.; Allen, Todd M.; Heath, Sonya L.; Goepfert, Paul; Bar, Katharine J.

2016-01-01

Background Identification of HIV-1 genomes responsible for establishing clinical infection in newly infected individuals is fundamental to prevention and pathogenesis research. Processing, storage, and transportation of the clinical samples required to perform these virologic assays in resource-limited settings requires challenging venipuncture and cold chain logistics. Here, we validate the use of dried-blood spots (DBS) as a simple and convenient alternative to collecting and storing frozen plasma. Methods We performed parallel nucleic acid extraction, single genome amplification (SGA), next generation sequencing (NGS), and phylogenetic analyses on plasma and DBS. Results We demonstrated the capacity to extract viral RNA from DBS and perform SGA to infer the complete nucleotide sequence of the transmitted/founder (TF) HIV-1 envelope gene and full-length genome in two acutely infected individuals. Using both SGA and NGS methodologies, we showed that sequences generated from DBS and plasma display comparable phylogenetic patterns in both acute and chronic infection. SGA was successful on samples with a range of plasma viremia, including samples as low as 1,700 copies/ml and an estimated ∼50 viral copies per blood spot. Further, we demonstrated reproducible efficiency in gp160 env sequencing in DBS stored at ambient temperature for up to three weeks or at -20°C for up to five months. Conclusions These findings support the use of DBS as a practical and cost-effective alternative to frozen plasma for clinical trials and translational research conducted in resource-limited settings. PMID:27819061

A Robust and Versatile Method of Combinatorial Chemical Synthesis of Gene Libraries via Hierarchical Assembly of Partially Randomized Modules

PubMed Central

Popova, Blagovesta; Schubert, Steffen; Bulla, Ingo; Buchwald, Daniela; Kramer, Wilfried

2015-01-01

A major challenge in gene library generation is to guarantee a large functional size and diversity that significantly increases the chances of selecting different functional protein variants. The use of trinucleotides mixtures for controlled randomization results in superior library diversity and offers the ability to specify the type and distribution of the amino acids at each position. Here we describe the generation of a high diversity gene library using tHisF of the hyperthermophile Thermotoga maritima as a scaffold. Combining various rational criteria with contingency, we targeted 26 selected codons of the thisF gene sequence for randomization at a controlled level. We have developed a novel method of creating full-length gene libraries by combinatorial assembly of smaller sub-libraries. Full-length libraries of high diversity can easily be assembled on demand from smaller and much less diverse sub-libraries, which circumvent the notoriously troublesome long-term archivation and repeated proliferation of high diversity ensembles of phages or plasmids. We developed a generally applicable software tool for sequence analysis of mutated gene sequences that provides efficient assistance for analysis of library diversity. Finally, practical utility of the library was demonstrated in principle by assessment of the conformational stability of library members and isolating protein variants with HisF activity from it. Our approach integrates a number of features of nucleic acids synthetic chemistry, biochemistry and molecular genetics to a coherent, flexible and robust method of combinatorial gene synthesis. PMID:26355961

Generation of Wheat Transcription Factor FOX Rice Lines and Systematic Screening for Salt and Osmotic Stress Tolerance.

PubMed

Wu, Jinxia; Zhang, Zhiguo; Zhang, Qian; Liu, Yayun; Zhu, Butuo; Cao, Jian; Li, Zhanpeng; Han, Longzhi; Jia, Jizeng; Zhao, Guangyao; Sun, Xuehui

2015-01-01

Transcription factors (TFs) play important roles in plant growth, development, and responses to environmental stress. In this study, we collected 1,455 full-length (FL) cDNAs of TFs, representing 45 families, from wheat and its relatives Triticum urartu, Aegilops speltoides, Aegilops tauschii, Triticum carthlicum, and Triticum aestivum. More than 15,000 T0 TF FOX (Full-length cDNA Over-eXpressing) rice lines were generated; of these, 10,496 lines set seeds. About 14.88% of the T0 plants showed obvious phenotypic changes. T1 lines (5,232 lines) were screened for salt and osmotic stress tolerance using 150 mM NaCl and 20% (v/v) PEG-4000, respectively. Among them, five lines (591, 746, 1647, 1812, and J4065) showed enhanced salt stress tolerance, five lines (591, 746, 898, 1078, and 1647) showed enhanced osmotic stress tolerance, and three lines (591, 746, and 1647) showed both salt and osmotic stress tolerance. Further analysis of the T-DNA flanking sequences showed that line 746 over-expressed TaEREB1, line 898 over-expressed TabZIPD, and lines 1812 and J4065 over-expressed TaOBF1a and TaOBF1b, respectively. The enhanced salt and osmotic stress tolerance of lines 898 and 1812 was confirmed by retransformation of the respective genes. Our results demonstrate that a heterologous FOX system may be used as an alternative genetic resource for the systematic functional analysis of the wheat genome.

A Robust and Versatile Method of Combinatorial Chemical Synthesis of Gene Libraries via Hierarchical Assembly of Partially Randomized Modules.

PubMed

Popova, Blagovesta; Schubert, Steffen; Bulla, Ingo; Buchwald, Daniela; Kramer, Wilfried

2015-01-01

A major challenge in gene library generation is to guarantee a large functional size and diversity that significantly increases the chances of selecting different functional protein variants. The use of trinucleotides mixtures for controlled randomization results in superior library diversity and offers the ability to specify the type and distribution of the amino acids at each position. Here we describe the generation of a high diversity gene library using tHisF of the hyperthermophile Thermotoga maritima as a scaffold. Combining various rational criteria with contingency, we targeted 26 selected codons of the thisF gene sequence for randomization at a controlled level. We have developed a novel method of creating full-length gene libraries by combinatorial assembly of smaller sub-libraries. Full-length libraries of high diversity can easily be assembled on demand from smaller and much less diverse sub-libraries, which circumvent the notoriously troublesome long-term archivation and repeated proliferation of high diversity ensembles of phages or plasmids. We developed a generally applicable software tool for sequence analysis of mutated gene sequences that provides efficient assistance for analysis of library diversity. Finally, practical utility of the library was demonstrated in principle by assessment of the conformational stability of library members and isolating protein variants with HisF activity from it. Our approach integrates a number of features of nucleic acids synthetic chemistry, biochemistry and molecular genetics to a coherent, flexible and robust method of combinatorial gene synthesis.

A Novel Videography Method for Generating Crack-Extension Resistance Curves in Small Bone Samples

PubMed Central

Katsamenis, Orestis L.; Jenkins, Thomas; Quinci, Federico; Michopoulou, Sofia; Sinclair, Ian; Thurner, Philipp J.

2013-01-01

Assessment of bone quality is an emerging solution for quantifying the effects of bone pathology or treatment. Perhaps one of the most important parameters characterising bone quality is the toughness behaviour of bone. Particularly, fracture toughness, is becoming a popular means for evaluating bone quality. The method is moving from a single value approach that models bone as a linear-elastic material (using the stress intensity factor, K) towards full crack extension resistance curves (R-curves) using a non-linear model (the strain energy release rate in J-R curves). However, for explanted human bone or small animal bones, there are difficulties in measuring crack-extension resistance curves due to size constraints at the millimetre and sub-millimetre scale. This research proposes a novel “whitening front tracking” method that uses videography to generate full fracture resistance curves in small bone samples where crack propagation cannot typically be observed. Here we present this method on sharp edge notched samples (<1 mm×1 mm×Length) prepared from four human femora tested in three-point bending. Each sample was loaded in a mechanical tester with the crack propagation recorded using videography and analysed using an algorithm to track the whitening (damage) zone. Using the “whitening front tracking” method, full R-curves and J-R curves could be generated for these samples. The curves for this antiplane longitudinal orientation were similar to those found in the literature, being between the published longitudinal and transverse orientations. The proposed technique shows the ability to generate full “crack” extension resistance curves by tracking the whitening front propagation to overcome the small size limitations and the single value approach. PMID:23405186

Microbes in the neonatal intensive care unit resemble those found in the gut of premature infants

PubMed Central

2014-01-01

Background The source inoculum of gastrointestinal tract (GIT) microbes is largely influenced by delivery mode in full-term infants, but these influences may be decoupled in very low birth weight (VLBW, <1,500 g) neonates via conventional broad-spectrum antibiotic treatment. We hypothesize the built environment (BE), specifically room surfaces frequently touched by humans, is a predominant source of colonizing microbes in the gut of premature VLBW infants. Here, we present the first matched fecal-BE time series analysis of two preterm VLBW neonates housed in a neonatal intensive care unit (NICU) over the first month of life. Results Fresh fecal samples were collected every 3 days and metagenomes sequenced on an Illumina HiSeq2000 device. For each fecal sample, approximately 33 swabs were collected from each NICU room from 6 specified areas: sink, feeding and intubation tubing, hands of healthcare providers and parents, general surfaces, and nurse station electronics (keyboard, mouse, and cell phone). Swabs were processed using a recently developed ‘expectation maximization iterative reconstruction of genes from the environment’ (EMIRGE) amplicon pipeline in which full-length 16S rRNA amplicons were sheared and sequenced using an Illumina platform, and short reads reassembled into full-length genes. Over 24,000 full-length 16S rRNA sequences were produced, generating an average of approximately 12,000 operational taxonomic units (OTUs) (clustered at 97% nucleotide identity) per room-infant pair. Dominant gut taxa, including Staphylococcus epidermidis, Klebsiella pneumoniae, Bacteroides fragilis, and Escherichia coli, were widely distributed throughout the room environment with many gut colonizers detected in more than half of samples. Reconstructed genomes from infant gut colonizers revealed a suite of genes that confer resistance to antibiotics (for example, tetracycline, fluoroquinolone, and aminoglycoside) and sterilizing agents, which likely offer a competitive advantage in the NICU environment. Conclusions We have developed a high-throughput culture-independent approach that integrates room surveys based on full-length 16S rRNA gene sequences with metagenomic analysis of fecal samples collected from infants in the room. The approach enabled identification of discrete ICU reservoirs of microbes that also colonized the infant gut and provided evidence for the presence of certain organisms in the room prior to their detection in the gut. PMID:24468033

Measuring Historical Coastal Change using GIS and the Change Polygon Approach

USGS Publications Warehouse

Smith, M.J.; Cromley, R.G.

2012-01-01

This study compares two automated approaches, the transect-from-baseline technique and a new change polygon method, for quantifying historical coastal change over time. The study shows that the transect-from-baseline technique is complicated by choice of a proper baseline as well as generating transects that intersect with each other rather than with the nearest shoreline. The change polygon method captures the full spatial difference between the positions of the two shorelines and average coastal change is the defined as the ratio of the net area divided by the shoreline length. Although then change polygon method is sensitive to the definition and measurement of shoreline length, the results are more invariant to parameter changes than the transect-from-baseline method, suggesting that the change polygon technique may be a more robust coastal change method. ?? 2012 Blackwell Publishing Ltd.

A high HIV-1 strain variability in London, UK, revealed by full-genome analysis: Results from the ICONIC project

PubMed Central

Frampton, Dan; Gallo Cassarino, Tiziano; Raffle, Jade; Hubb, Jonathan; Ferns, R. Bridget; Waters, Laura; Tong, C. Y. William; Kozlakidis, Zisis; Hayward, Andrew; Kellam, Paul; Pillay, Deenan; Clark, Duncan; Nastouli, Eleni; Leigh Brown, Andrew J.

2018-01-01

Background & methods The ICONIC project has developed an automated high-throughput pipeline to generate HIV nearly full-length genomes (NFLG, i.e. from gag to nef) from next-generation sequencing (NGS) data. The pipeline was applied to 420 HIV samples collected at University College London Hospitals NHS Trust and Barts Health NHS Trust (London) and sequenced using an Illumina MiSeq at the Wellcome Trust Sanger Institute (Cambridge). Consensus genomes were generated and subtyped using COMET, and unique recombinants were studied with jpHMM and SimPlot. Maximum-likelihood phylogenetic trees were constructed using RAxML to identify transmission networks using the Cluster Picker. Results The pipeline generated sequences of at least 1Kb of length (median = 7.46Kb, IQR = 4.01Kb) for 375 out of the 420 samples (89%), with 174 (46.4%) being NFLG. A total of 365 sequences (169 of them NFLG) corresponded to unique subjects and were included in the down-stream analyses. The most frequent HIV subtypes were B (n = 149, 40.8%) and C (n = 77, 21.1%) and the circulating recombinant form CRF02_AG (n = 32, 8.8%). We found 14 different CRFs (n = 66, 18.1%) and multiple URFs (n = 32, 8.8%) that involved recombination between 12 different subtypes/CRFs. The most frequent URFs were B/CRF01_AE (4 cases) and A1/D, B/C, and B/CRF02_AG (3 cases each). Most URFs (19/26, 73%) lacked breakpoints in the PR+RT pol region, rendering them undetectable if only that was sequenced. Twelve (37.5%) of the URFs could have emerged within the UK, whereas the rest were probably imported from sub-Saharan Africa, South East Asia and South America. For 2 URFs we found highly similar pol sequences circulating in the UK. We detected 31 phylogenetic clusters using the full dataset: 25 pairs (mostly subtypes B and C), 4 triplets and 2 quadruplets. Some of these were not consistent across different genes due to inter- and intra-subtype recombination. Clusters involved 70 sequences, 19.2% of the dataset. Conclusions The initial analysis of genome sequences detected substantial hidden variability in the London HIV epidemic. Analysing full genome sequences, as opposed to only PR+RT, identified previously undetected recombinants. It provided a more reliable description of CRFs (that would be otherwise misclassified) and transmission clusters. PMID:29389981

Multi Scale Modeling of Continuous Aramid Fiber Reinforced Polymer Matrix Composites Used in Ballistic Protection Applications

DTIC Science & Technology

2014-11-16

related to identification of the type and the extent of data generated at a finer length scale to the adjacent coarser length scale, as well as seamless ...data generated at a finer length scale to the adjacent coarser length scale, as well as seamless integration of different length scales into a unified...composite laminate consisting of 32 laminae and impacted (at a 0° obliquity angle and an incident velocity of 500 m/s) by a 0.30 caliber steel

Three Immunoproteasome-Associated Subunits Cooperatively Generate a Cytotoxic T-Lymphocyte Epitope of Epstein-Barr Virus LMP2A by Overcoming Specific Structures Resistant to Epitope Liberation

PubMed Central

Ito, Yoshinori; Kondo, Eisei; Demachi-Okamura, Ayako; Akatsuka, Yoshiki; Tsujimura, Kunio; Tanimoto, Mitsune; Morishima, Yasuo; Takahashi, Toshitada; Kuzushima, Kiyotaka

2006-01-01

The precise roles of gamma interferon-inducible immunoproteasome-associated molecules in generation of cytotoxic T-lymphocyte (CTL) epitopes have yet to be fully elucidated. We describe here a unique epitope derived from the Epstein-Barr virus (EBV) latent membrane protein 2A (LMP2A) presented by HLA-A*2402 molecules. Generation of the epitope, designated LMP2A222-230, from the full-length protein requires the immunoproteasome subunit low-molecular-weight protein 7 (ip-LMP7) and the proteasome activator 28-α subunit and is accelerated by ip-LMP2, as revealed by gene expression experiments using an LMP2A222-230-specific CTL clone as a responder in enzyme-linked immunospot assays. The unequivocal involvement of all three components was confirmed by RNA interference gene silencing. Interestingly, the LMP2A222-230 epitope could be efficiently generated from incomplete EBV-LMP2A fragments that were produced by puromycin treatment or gene-engineered shortened EBV-LMP2A lacking some of its hydrophobic domains. In addition, epitope generation was increased by a single amino acid substitution from leucine to alanine immediately flanking the C terminus, this being predicted by a web-accessible program to increase the cleavage strength. Taken together, the data indicate that the generation of LMP2A222-230 is influenced not only by extrinsic factors such as immunoproteasomes but also by intrinsic factors such as the length of the EBV-LMP2A protein and proteasomal cleavage strength at specific positions in the source antigen. PMID:16378990

The Staphylococcus aureus extracellular adherence protein (Eap) adopts an elongated but structured conformation in solution.

PubMed

Hammel, Michal; Nemecek, Daniel; Keightley, J Andrew; Thomas, George J; Geisbrecht, Brian V

2007-12-01

The extracellular adherence protein (Eap) of Staphylococcus aureus participates in a wide range of protein-protein interactions that facilitate the initiation and dissemination of Staphylococcal disease. In this report, we describe the use of a multidisciplinary approach to characterize the solution structure of full-length Eap. In contrast to previous reports suggesting that a six-domain isoform of Eap undergoes multimerization, sedimentation equilibrium analytical ultracentrifugation data revealed that a four-domain isoform of Eap is a monomer in solution. In vitro proteolysis and solution small angle X-ray scattering studies both indicate that Eap adopts an extended conformation in solution, where the linkers connecting sequential EAP modules are solvent exposed. Construction of a low-resolution model of full-length Eap using a combination of ab initio deconvolution of the SAXS data and rigid body modeling of the EAP domain crystal structure suggests that full-length Eap may present several unique concave surfaces capable of participating in ligand binding. These results also raise the possibility that such surfaces may be held together by additional interactions between adjacent EAP modules. This hypothesis is supported by a comparative Raman spectroscopic analysis of full-length Eap and a stoichiometric solution of the individual EAP modules, which indicates the presence of additional secondary structure and a greater extent of hydrogen/deuterium exchange protection in full-length Eap. Our results provide the first insight into the solution structure of full-length Eap and an experimental basis for interpreting the EAP domain crystal structures within the context of the full-length molecule. They also lay a foundation for future studies into the structural and molecular bases of Eap-mediated protein-protein interactions with its many ligands.

The Staphylococcus aureus extracellular adherence protein (Eap) adopts an elongated but structured conformation in solution

PubMed Central

Hammel, Michal; Němeček, Daniel; Keightley, J. Andrew; Thomas, George J.; Geisbrecht, Brian V.

2007-01-01

The extracellular adherence protein (Eap) of Staphylococcus aureus participates in a wide range of protein–protein interactions that facilitate the initiation and dissemination of Staphylococcal disease. In this report, we describe the use of a multidisciplinary approach to characterize the solution structure of full-length Eap. In contrast to previous reports suggesting that a six-domain isoform of Eap undergoes multimerization, sedimentation equilibrium analytical ultracentrifugation data revealed that a four-domain isoform of Eap is a monomer in solution. In vitro proteolysis and solution small angle X-ray scattering studies both indicate that Eap adopts an extended conformation in solution, where the linkers connecting sequential EAP modules are solvent exposed. Construction of a low-resolution model of full-length Eap using a combination of ab initio deconvolution of the SAXS data and rigid body modeling of the EAP domain crystal structure suggests that full-length Eap may present several unique concave surfaces capable of participating in ligand binding. These results also raise the possibility that such surfaces may be held together by additional interactions between adjacent EAP modules. This hypothesis is supported by a comparative Raman spectroscopic analysis of full-length Eap and a stoichiometric solution of the individual EAP modules, which indicates the presence of additional secondary structure and a greater extent of hydrogen/deuterium exchange protection in full-length Eap. Our results provide the first insight into the solution structure of full-length Eap and an experimental basis for interpreting the EAP domain crystal structures within the context of the full-length molecule. They also lay a foundation for future studies into the structural and molecular bases of Eap-mediated protein–protein interactions with its many ligands. PMID:18029416

Generation and Analysis of a Large-Scale Expressed Sequence Tag Database from a Full-Length Enriched cDNA Library of Developing Leaves of Gossypium hirsutum L

PubMed Central

Pang, Chaoyou; Fan, Shuli; Song, Meizhen; Yu, Shuxun

2013-01-01

Background Cotton (Gossypium hirsutum L.) is one of the world’s most economically-important crops. However, its entire genome has not been sequenced, and limited resources are available in GenBank for understanding the molecular mechanisms underlying leaf development and senescence. Methodology/Principal Findings In this study, 9,874 high-quality ESTs were generated from a normalized, full-length cDNA library derived from pooled RNA isolated from throughout leaf development during the plant blooming stage. After clustering and assembly of these ESTs, 5,191 unique sequences, representative 1,652 contigs and 3,539 singletons, were obtained. The average unique sequence length was 682 bp. Annotation of these unique sequences revealed that 84.4% showed significant homology to sequences in the NCBI non-redundant protein database, and 57.3% had significant hits to known proteins in the Swiss-Prot database. Comparative analysis indicated that our library added 2,400 ESTs and 991 unique sequences to those known for cotton. The unigenes were functionally characterized by gene ontology annotation. We identified 1,339 and 200 unigenes as potential leaf senescence-related genes and transcription factors, respectively. Moreover, nine genes related to leaf senescence and eleven MYB transcription factors were randomly selected for quantitative real-time PCR (qRT-PCR), which revealed that these genes were regulated differentially during senescence. The qRT-PCR for three GhYLSs revealed that these genes express express preferentially in senescent leaves. Conclusions/Significance These EST resources will provide valuable sequence information for gene expression profiling analyses and functional genomics studies to elucidate their roles, as well as for studying the mechanisms of leaf development and senescence in cotton and discovering candidate genes related to important agronomic traits of cotton. These data will also facilitate future whole-genome sequence assembly and annotation in G. hirsutum and comparative genomics among Gossypium species. PMID:24146870

Large-Scale Collection and Analysis of Full-Length cDNAs from Brachypodium distachyon and Integration with Pooideae Sequence Resources

PubMed Central

Mochida, Keiichi; Uehara-Yamaguchi, Yukiko; Takahashi, Fuminori; Yoshida, Takuhiro; Sakurai, Tetsuya; Shinozaki, Kazuo

2013-01-01

A comprehensive collection of full-length cDNAs is essential for correct structural gene annotation and functional analyses of genes. We constructed a mixed full-length cDNA library from 21 different tissues of Brachypodium distachyon Bd21, and obtained 78,163 high quality expressed sequence tags (ESTs) from both ends of ca. 40,000 clones (including 16,079 contigs). We updated gene structure annotations of Brachypodium genes based on full-length cDNA sequences in comparison with the latest publicly available annotations. About 10,000 non-redundant gene models were supported by full-length cDNAs; ca. 6,000 showed some transcription unit modifications. We also found ca. 580 novel gene models, including 362 newly identified in Bd21. Using the updated transcription start sites, we searched a total of 580 plant cis-motifs in the −3 kb promoter regions and determined a genome-wide Brachypodium promoter architecture. Furthermore, we integrated the Brachypodium full-length cDNAs and updated gene structures with available sequence resources in wheat and barley in a web-accessible database, the RIKEN Brachypodium FL cDNA database. The database represents a “one-stop” information resource for all genomic information in the Pooideae, facilitating functional analysis of genes in this model grass plant and seamless knowledge transfer to the Triticeae crops. PMID:24130698

Separated-orbit bisected energy-recovered linear accelerator

DOEpatents

Douglas, David R.

2015-09-01

A separated-orbit bisected energy-recovered linear accelerator apparatus and method. The accelerator includes a first linac, a second linac, and a plurality of arcs of differing path lengths, including a plurality of up arcs, a plurality of downgoing arcs, and a full energy arc providing a path independent of the up arcs and downgoing arcs. The up arcs have a path length that is substantially a multiple of the RF wavelength and the full energy arc includes a path length that is substantially an odd half-integer multiple of the RF wavelength. Operation of the accelerator includes accelerating the beam utilizing the linacs and up arcs until the beam is at full energy, at full energy executing a full recirculation to the second linac using a path length that is substantially an odd half-integer of the RF wavelength, and then decelerating the beam using the linacs and downgoing arcs.

Macaca specific exon creation event generates a novel ZKSCAN5 transcript.

PubMed

Kim, Young-Hyun; Choe, Se-Hee; Song, Bong-Seok; Park, Sang-Je; Kim, Myung-Jin; Park, Young-Ho; Yoon, Seung-Bin; Lee, Youngjeon; Jin, Yeung Bae; Sim, Bo-Woong; Kim, Ji-Su; Jeong, Kang-Jin; Kim, Sun-Uk; Lee, Sang-Rae; Park, Young-Il; Huh, Jae-Won; Chang, Kyu-Tae

2016-02-15

ZKSCAN5 (also known as ZFP95) is a zinc-finger protein belonging to the Krűppel family. ZKSCAN5 contains a SCAN box and a KRAB A domain and is proposed to play a distinct role during spermatogenesis. In humans, alternatively spliced ZKSCAN5 transcripts with different 5'-untranslated regions (UTRs) have been identified. However, investigation of our Macaca UniGene Database revealed novel alternative ZKSCAN5 transcripts that arose due to an exon creation event. Therefore, in this study, we identified the full-length sequences of ZKSCAN5 and its alternative transcripts in Macaca spp. Additionally, we investigated different nonhuman primate sequences to elucidate the molecular mechanism underlying the exon creation event. We analyzed the evolutionary features of the ZKSCAN5 transcripts by reverse transcription polymerase chain reaction (RT-PCR) and genomic PCR, and by sequencing various nonhuman primate DNA and RNA samples. The exon-created transcript was only detected in the Macaca lineage (crab-eating monkey and rhesus monkey). Full-length sequence analysis by rapid amplification of cDNA ends (RACE) identified ten full-length transcripts and four functional isoforms of ZKSCAN5. Protein sequence analyses revealed the presence of two groups of isoforms that arose because of differences in start-codon usage. Together, our results demonstrate that there has been specific selection for a discrete set of ZKSCAN5 variants in the Macaca lineage. Furthermore, study of this locus (and perhaps others) in Macaca spp. might facilitate our understanding of the evolutionary pressures that have shaped the mechanism of exon creation in primates. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

Using Optimal Test Assembly Methods for Shortening Patient-Reported Outcome Measures: Development and Validation of the Cochin Hand Function Scale-6: A Scleroderma Patient-Centered Intervention Network Cohort Study.

PubMed

Levis, Alexander W; Harel, Daphna; Kwakkenbos, Linda; Carrier, Marie-Eve; Mouthon, Luc; Poiraudeau, Serge; Bartlett, Susan J; Khanna, Dinesh; Malcarne, Vanessa L; Sauve, Maureen; van den Ende, Cornelia H M; Poole, Janet L; Schouffoer, Anne A; Welling, Joep; Thombs, Brett D

2016-11-01

To develop and validate a short form of the Cochin Hand Function Scale (CHFS), which measures hand disability, for use in systemic sclerosis, using objective criteria and reproducible techniques. Responses on the 18-item CHFS were obtained from English-speaking patients enrolled in the Scleroderma Patient-Centered Intervention Network Cohort. CHFS unidimensionality was verified using confirmatory factor analysis, and an item response theory model was fit to CHFS items. Optimal test assembly (OTA) methods identified a maximally precise short form for each possible form length between 1 and 17 items. The final short form selected was the form with the least number of items that maintained statistically equivalent convergent validity, compared to the full-length CHFS, with the Health Assessment Questionnaire (HAQ) disability index (DI) and the physical function domain of the 29-item Patient-Reported Outcomes Measurement Information System (PROMIS-29). There were 601 patients included. A 6-item short form of the CHFS (CHFS-6) was selected. The CHFS-6 had a Cronbach's alpha of 0.93. Correlations of the CHFS-6 summed score with HAQ DI (r = 0.79) and PROMIS-29 physical function (r = -0.54) were statistically equivalent to the CHFS (r = 0.81 and r = -0.56). The correlation with the full CHFS was high (r = 0.98). The OTA procedure generated a valid short form of the CHFS with minimal loss of information compared to the full-length form. The OTA method used was based on objective, prespecified criteria, but should be further studied for viability as a general procedure for shortening patient-reported outcome measures in health research. © 2016, American College of Rheumatology.

Mini-DNA barcode in identification of the ornamental fish: A case study from Northeast India.

PubMed

Dhar, Bishal; Ghosh, Sankar Kumar

2017-09-05

The ornamental fishes were exported under the trade names or generic names, thus creating problems in species identification. In this regard, DNA barcoding could effectively elucidate the actual species status. However, the problem arises if the specimen is having taxonomic disputes, falsified by trade/generic names, etc., On the other hand, barcoding the archival museum specimens would be of greater benefit to address such issues as it would create firm, error-free reference database for rapid identification of any species. This can be achieved only by generating short sequences as DNA from chemically preserved are mostly degraded. Here we aimed to identify a short stretch of informative sites within the full-length barcode segment, capable of delineating diverse group of ornamental fish species, commonly traded from NE India. We analyzed 287 full-length barcode sequences from the major fish orders and compared the interspecific K2P distance with nucleotide substitutions patterns and found a strong correlation of interspecies distance with transversions (0.95, p<0.001). We, therefore, proposed a short stretch of 171bp (transversion rich) segment as mini-barcode. The proposed segment was compared with the full-length barcodes and found to delineate the species effectively. Successful PCR amplification and sequencing of the 171bp segment using designed primers for different orders validated it as mini-barcodes for ornamental fishes. Thus, our findings would be helpful in strengthening the global database with the sequence of archived fish species as well as an effective identification tool of the traded ornamental fish species, as a less time consuming, cost effective field-based application. Copyright © 2017 Elsevier B.V. All rights reserved.

Fluorescent IgG fusion proteins made in E. coli

PubMed Central

Luria, Yael; Raichlin, Dina; Benhar, Itai

2012-01-01

Antibodies are among the most powerful tools in biological and biomedical research and are presently the fastest growing category of new bio-pharmaceutics. The most common format of antibody applied for therapeutic, diagnostic and analytical purposes is the IgG format. For medical applications, recombinant IgGs are made in cultured mammalian cells in a process that is too expensive to be considered for producing antibodies for diagnostic and analytical purposes. Therefore, for such purposes, mouse monoclonal antibodies or polyclonal sera from immunized animals are used. While looking for an easier and more rapid way to prepare full-length IgGs for therapeutic purposes, we recently developed and reported an expression and purification protocol for full-length IgGs, and IgG-based fusion proteins in E. coli, called “Inclonals.” By applying the Inclonals technology, we could generate full-length IgGs that are genetically fused to toxins. The aim of the study described herein was to evaluate the possibility of applying the “Inclonals” technology for preparing IgG-fluorophore fusion proteins. We found that IgG fused to the green fluorescent proteins enhanced GFP (EGFP) while maintaining functionality in binding, lost most of its fluorescence during the refolding process. In contrast, we found that green fluorescent Superfolder GFP (SFGFP)-fused IgG and red fluorescent mCherry-fused IgG were functional in antigen binding and maintained fluorescence intensity. In addition, we found that we can link several SFGFPs in tandem to each IgG, with fluorescence intensity increasing accordingly. Fluorescent IgGs made in E. coli may become attractive alternatives to monoclonal or polyclonal fluorescent antibodies derived from animals. PMID:22531449

Genetically encoded photocross-linkers determine the biological binding site of exendin-4 peptide in the N-terminal domain of the intact human glucagon-like peptide-1 receptor (GLP-1R)

PubMed Central

Koole, Cassandra; Reynolds, Christopher A.; Mobarec, Juan C.; Hick, Caroline; Sexton, Patrick M.; Sakmar, Thomas P.

2017-01-01

The glucagon-like peptide-1 receptor (GLP-1R) is a key therapeutic target in the management of type II diabetes mellitus, with actions including regulation of insulin biosynthesis and secretion, promotion of satiety, and preservation of β-cell mass. Like most class B G protein-coupled receptors (GPCRs), there is limited knowledge linking biological activity of the GLP-1R with the molecular structure of an intact, full-length, and functional receptor·ligand complex. In this study, we have utilized genetic code expansion to site-specifically incorporate the photoactive amino acid p-azido-l-phenylalanine (azF) into N-terminal residues of a full-length functional human GLP-1R in mammalian cells. UV-mediated photolysis of azF was then carried out to induce targeted photocross-linking to determine the proximity of the azido group in the mutant receptor with the peptide exendin-4. Cross-linking data were compared directly with the crystal structure of the isolated N-terminal extracellular domain of the GLP-1R in complex with exendin(9–39), revealing both similarities as well as distinct differences in the mode of interaction. Generation of a molecular model to accommodate the photocross-linking constraints highlights the potential influence of environmental conditions on the conformation of the receptor·peptide complex, including folding dynamics of the peptide and formation of dimeric and higher order oligomeric receptor multimers. These data demonstrate that crystal structures of isolated receptor regions may not give a complete reflection of peptide/receptor interactions and should be combined with additional experimental constraints to reveal peptide/receptor interactions occurring in the dynamic, native, and full-length receptor state. PMID:28283573

Genome-wide comparisons of phylogenetic similarities between partial genomic regions and the full-length genome in Hepatitis E virus genotyping.

PubMed

Wang, Shuai; Wei, Wei; Luo, Xuenong; Cai, Xuepeng

2014-01-01

Besides the complete genome, different partial genomic sequences of Hepatitis E virus (HEV) have been used in genotyping studies, making it difficult to compare the results based on them. No commonly agreed partial region for HEV genotyping has been determined. In this study, we used a statistical method to evaluate the phylogenetic performance of each partial genomic sequence from a genome wide, by comparisons of evolutionary distances between genomic regions and the full-length genomes of 101 HEV isolates to identify short genomic regions that can reproduce HEV genotype assignments based on full-length genomes. Several genomic regions, especially one genomic region at the 3'-terminal of the papain-like cysteine protease domain, were detected to have relatively high phylogenetic correlations with the full-length genome. Phylogenetic analyses confirmed the identical performances between these regions and the full-length genome in genotyping, in which the HEV isolates involved could be divided into reasonable genotypes. This analysis may be of value in developing a partial sequence-based consensus classification of HEV species.

A combined computational and structural model of the full-length human prolactin receptor

PubMed Central

Bugge, Katrine; Papaleo, Elena; Haxholm, Gitte W.; Hopper, Jonathan T. S.; Robinson, Carol V.; Olsen, Johan G.; Lindorff-Larsen, Kresten; Kragelund, Birthe B.

2016-01-01

The prolactin receptor is an archetype member of the class I cytokine receptor family, comprising receptors with fundamental functions in biology as well as key drug targets. Structurally, each of these receptors represent an intriguing diversity, providing an exceptionally challenging target for structural biology. Here, we access the molecular architecture of the monomeric human prolactin receptor by combining experimental and computational efforts. We solve the NMR structure of its transmembrane domain in micelles and collect structural data on overlapping fragments of the receptor with small-angle X-ray scattering, native mass spectrometry and NMR spectroscopy. Along with previously published data, these are integrated by molecular modelling to generate a full receptor structure. The result provides the first full view of a class I cytokine receptor, exemplifying the architecture of more than 40 different receptor chains, and reveals that the extracellular domain is merely the tip of a molecular iceberg. PMID:27174498

Characterization of full-length MHC class II sequences in Indonesian and Vietnamese cynomolgus macaques.

PubMed

Creager, Hannah M; Becker, Ericka A; Sandman, Kelly K; Karl, Julie A; Lank, Simon M; Bimber, Benjamin N; Wiseman, Roger W; Hughes, Austin L; O'Connor, Shelby L; O'Connor, David H

2011-09-01

In recent years, the use of cynomolgus macaques in biomedical research has increased greatly. However, with the exception of the Mauritian population, knowledge of the MHC class II genetics of the species remains limited. Here, using cDNA cloning and Sanger sequencing, we identified 127 full-length MHC class II alleles in a group of 12 Indonesian and 12 Vietnamese cynomolgus macaques. Forty two of these were completely novel to cynomolgus macaques while 61 extended the sequence of previously identified alleles from partial to full length. This more than doubles the number of full-length cynomolgus macaque MHC class II alleles available in GenBank, significantly expanding the allele library for the species and laying the groundwork for future evolutionary and functional studies.

Autocorrelation peaks in congruential pseudorandom number generators

NASA Technical Reports Server (NTRS)

Neuman, F.; Merrick, R. B.

1976-01-01

The complete correlation structure of several congruential pseudorandom number generators (PRNG) of the same type and small cycle length was studied to deal with the problem of congruential PRNG almost repeating themselves at intervals smaller than their cycle lengths, during simulation of bandpass filtered normal random noise. Maximum period multiplicative and mixed congruential generators were studied, with inferences drawn from examination of several tractable members of a class of random number generators, and moduli from 2 to the 5th power to 2 to the 9th power. High correlation is shown to exist in mixed and multiplicative congruential random number generators and prime moduli Lehmer generators for shifts a fraction of their cycle length. The random noise sequences in question are required when simulating electrical noise, air turbulence, or time variation of wind parameters.

Statistical properties of filtered pseudorandom digital sequences formed from the sum of maximum-length sequences

NASA Technical Reports Server (NTRS)

Wallace, G. R.; Weathers, G. D.; Graf, E. R.

1973-01-01

The statistics of filtered pseudorandom digital sequences called hybrid-sum sequences, formed from the modulo-two sum of several maximum-length sequences, are analyzed. The results indicate that a relation exists between the statistics of the filtered sequence and the characteristic polynomials of the component maximum length sequences. An analysis procedure is developed for identifying a large group of sequences with good statistical properties for applications requiring the generation of analog pseudorandom noise. By use of the analysis approach, the filtering process is approximated by the convolution of the sequence with a sum of unit step functions. A parameter reflecting the overall statistical properties of filtered pseudorandom sequences is derived. This parameter is called the statistical quality factor. A computer algorithm to calculate the statistical quality factor for the filtered sequences is presented, and the results for two examples of sequence combinations are included. The analysis reveals that the statistics of the signals generated with the hybrid-sum generator are potentially superior to the statistics of signals generated with maximum-length generators. Furthermore, fewer calculations are required to evaluate the statistics of a large group of hybrid-sum generators than are required to evaluate the statistics of the same size group of approximately equivalent maximum-length sequences.

Observation of a rainbow of visible colors in a near infrared cascaded Raman fiber laser and its novel application as a diagnostic tool for length resolved spectral analysis

NASA Astrophysics Data System (ADS)

Aparanji, Santosh; Balaswamy, V.; Arun, S.; Supradeepa, V. R.

2018-02-01

In this work, we report and analyse the surprising observation of a rainbow of visible colors, spanning 390nm to 620nm, in silica-based, Near Infrared, continuous-wave, cascaded Raman fiber lasers. The cascaded Raman laser is pumped at 1117nm at around 200W and at full power we obtain 100 W at 1480nm. With increasing pump power at 1117nm, the fiber constituting the Raman laser glows in various hues along its length. From spectroscopic analysis of the emitted visible light, it was identified to be harmonic and sum-frequency components of various locally propagating wavelength components. In addition to third harmonic components, surprisingly, even 2nd harmonic components were observed. Despite being a continuous-wave laser, we expect the phase-matching occurring between the core-propagating NIR light with the cladding-propagating visible wavelengths and the intensity fluctuations characteristic of Raman lasers to have played a major role in generation of visible light. In addition, this surprising generation of visible light provides us a powerful non-contact method to deduce the spectrum of light propagating in the fiber. Using static images of the fiber captured by a standard visible camera such as a DSLR, we demonstrate novel, image-processing based techniques to deduce the wavelength component propagating in the fiber at any given spatial location. This provides a powerful diagnostic tool for both length and power resolved spectral analysis in Raman fiber lasers. This helps accurate prediction of the optimal length of fiber required for complete and efficient conversion to a given Stokes wavelength.

Virtual hybrid test control of sinuous crack

NASA Astrophysics Data System (ADS)

Jailin, Clément; Carpiuc, Andreea; Kazymyrenko, Kyrylo; Poncelet, Martin; Leclerc, Hugo; Hild, François; Roux, Stéphane

2017-05-01

The present study aims at proposing a new generation of experimental protocol for analysing crack propagation in quasi brittle materials. The boundary conditions are controlled in real-time to conform to a predefined crack path. Servo-control is achieved through a full-field measurement technique to determine the pre-set fracture path and a simple predictor model based on linear elastic fracture mechanics to prescribe the boundary conditions on the fly so that the actual crack path follows at best the predefined trajectory. The final goal is to identify, for instance, non-local damage models involving internal lengths. The validation of this novel procedure is performed via a virtual test-case based on an enriched damage model with an internal length scale, a prior chosen sinusoidal crack path and a concrete sample. Notwithstanding the fact that the predictor model selected for monitoring the test is a highly simplified picture of the targeted constitutive law, the proposed protocol exhibits a much improved sensitivity to the sought parameters such as internal lengths as assessed from the comparison with other available experimental tests.

A novel murine allele of Intraflagellar Transport Protein 172 causes a syndrome including VACTERL-like features with hydrocephalus.

PubMed

Friedland-Little, Joshua M; Hoffmann, Andrew D; Ocbina, Polloneal Jymmiel R; Peterson, Mike A; Bosman, Joshua D; Chen, Yan; Cheng, Steven Y; Anderson, Kathryn V; Moskowitz, Ivan P

2011-10-01

The primary cilium is emerging as a crucial regulator of signaling pathways central to vertebrate development and human disease. We identified atrioventricular canal 1 (avc1), a mouse mutation that caused VACTERL association with hydrocephalus, or VACTERL-H. We showed that avc1 is a hypomorphic mutation of intraflagellar transport protein 172 (Ift172), required for ciliogenesis and Hedgehog (Hh) signaling. Phenotypically, avc1 caused VACTERL-H but not abnormalities in left-right (L-R) axis formation. Avc1 resulted in structural cilia defects, including truncated cilia in vivo and in vitro. We observed a dose-dependent requirement for Ift172 in ciliogenesis using an allelic series generated with Ift172(avc1) and Ift172(wim), an Ift172 null allele: cilia were present on 42% of avc1 mouse embryonic fibroblast (MEF) and 28% of avc1/wim MEFs, in contrast to >90% of wild-type MEFs. Furthermore, quantitative cilium length analysis identified two specific cilium populations in mutant MEFS: a normal population with normal IFT and a truncated population, 50% of normal length, with disrupted IFT. Cells from wild-type embryos had predominantly full-length cilia, avc1 embryos, with Hh signaling abnormalities but not L-R abnormalities, had cilia equally divided between full-length and truncated, and avc1/wim embryos, with both Hh signaling and L-R abnormalities, were primarily truncated. Truncated Ift172 mutant cilia showed defects of the distal ciliary axoneme, including disrupted IFT88 localization and Hh-dependent Gli2 localization. We propose a model in which mutation of Ift172 results in a specific class of abnormal cilia, causing disrupted Hh signaling while maintaining L-R axis determination, and resulting in the VACTERL-H phenotype.

Generation and Characterization of HIV-1 Transmitted and Founder Virus Consensus Sequence from Intravenous Drug Users in Xinjiang, China.

PubMed

Li, Fan; Ma, Liying; Feng, Yi; Hu, Jing; Ni, Na; Ruan, Yuhua; Shao, Yiming

2017-06-01

HIV-1 transmission in intravenous drug users (IDUs) has been characterized by high genetic multiplicity and suggests a greater challenge for HIV-1 infection blocking. We investigated a total of 749 sequences of full-length gp160 gene obtained by single genome sequencing (SGS) from 22 HIV-1 early infected IDUs in Xinjiang province, northwest China, and generated a transmitted and founder virus (T/F virus) consensus sequence (IDU.CON). The T/F virus was classified as subtype CRF07_BC and predicted to be CCR5-tropic virus. The variable region (V1, V2, and V4 loop) of IDU.CON showed length variation compared with the heterosexual T/F virus consensus sequence (HSX.CON) and homosexual T/F virus consensus sequence (MSM.CON). A total of 26 N-linked glycosylation sites were discovered in the IDU.CON sequence, which is less than that of MSM.CON and HSX.CON. Characterization of T/F virus from IDUs highlights the genetic make-up and complexity of virus near the moment of transmission or in early infection preceding systemic dissemination and is important toward the development of an effective HIV-1 preventive methods, including vaccines.

LIAD-fs scheme for studies of ultrafast laser interactions with gas phase biomolecules.

PubMed

Calvert, C R; Belshaw, L; Duffy, M J; Kelly, O; King, R B; Smyth, A G; Kelly, T J; Costello, J T; Timson, D J; Bryan, W A; Kierspel, T; Rice, P; Turcu, I C E; Cacho, C M; Springate, E; Williams, I D; Greenwood, J B

2012-05-14

Laser induced acoustic desorption (LIAD) has been used for the first time to study the parent ion production and fragmentation mechanisms of a biological molecule in an intense femtosecond (fs) laser field. The photoacoustic shock wave generated in the analyte substrate (thin Ta foil) has been simulated using the hydrodynamic HYADES code, and the full LIAD process has been experimentally characterised as a function of the desorption UV-laser pulse parameters. Observed neutral plumes of densities >10(9) cm(-3) which are free from solvent or matrix contamination demonstrate the suitability and potential of the source for studying ultrafast dynamics in the gas phase using fs laser pulses. Results obtained with phenylalanine show that through manipulation of fundamental femtosecond laser parameters (such as pulse length, intensity and wavelength), energy deposition within the molecule can be controlled to allow enhancement of parent ion production or generation of characteristic fragmentation patterns. In particular by reducing the pulse length to a timescale equivalent to the fastest vibrational periods in the molecule, we demonstrate how fragmentation of the molecule can be minimised whilst maintaining a high ionisation efficiency. This journal is © the Owner Societies 2012

Development of the Flight Tether for ProSEDS

NASA Technical Reports Server (NTRS)

Curtis, Leslie; Vaughn, Jason; Welzyn, Ken; Carroll, Joe; Brown, Norman S. (Technical Monitor)

2002-01-01

The Propulsive Small Expendable Deployer System (ProSEDS) space experiment will demonstrate the use of an electrodynamic tether propulsion system to generate thrust in space by decreasing the orbital altitude of a Delta 11 Expendable Launch Vehicle second stage. ProSEDS will use the flight-proven Small Expendable Deployer System to deploy a newly designed and developed tether which will provide tether generated drag thrust of approx. 0.4 N. The development and production of very long tethers with specific properties for performance and survivability will be required to enable future tether missions. The ProSEDS tether design and the development process may provide some lessons learned for these future missions. The ProSEDS system requirements drove the design of the tether to have three different sections of tether each serving a specialized purpose. The tether is a total of 15 kilometers long: 10 kilometers of a non-conductive Dyneema lead tether; 5 km of CCOR conductive coated wire; and 220 meters of insulated wire with a protective Kevlar overbraid. Production and joining of long tether lengths involved many development efforts. Extensive testing of tether materials including ground deployment of the full-length ProSEDS tether was conducted to validate the tether design and performance before flight.

The full-length microRNA cluster in the intron of large latency transcript is associated with the virulence of pseudorabies virus.

PubMed

Wang, Xin; Zhang, Mei-Mei; Yan, Kai; Tang, Qi; Wu, Yi-Quan; He, Wen-Bo; Chen, Huan-Chun; Liu, Zheng-Fei

2018-07-01

Pseudorabies virus (PRV), the etiological pathogen of Aujeszky's disease, belongs to the Alphaherpesvirus subfamily. Large latency transcript (LLT), the most abundant PRV transcript, harbors a ~ 4.6 kb microRNA (miRNA) cluster-encoding intron. To investigate the function of the LLT miRNA cluster during the life cycle of PRV, we generated a miRNA cluster mutation virus (PRV-∆miR cluster) and revertant virus. Analysis of the growth kinetics of PRV-ΔmiR cluster-infected cells revealed significantly smaller plaques and lower titers than the wild-type and revertant viruses. The mutation virus exhibited increased IE180 and decreased EP0 expression. The clinical symptoms observed in mice infected with PRV-ΔmiR cluster revealed that the miRNA cluster is involved in the pathogenesis of PRV. Physical parameters, virus shedding assays, and the SN 50 titers revealed that the miRNA cluster enhances PRV virulence in pigs. Collectively, our findings suggest that the full-length miRNA cluster is involved in PRV replication and virulence. Copyright © 2018 Elsevier Inc. All rights reserved.

Cross-cultural estimation of the human generation interval for use in genetics-based population divergence studies.

PubMed

Fenner, Jack N

2005-10-01

The length of the human generation interval is a key parameter when using genetics to date population divergence events. However, no consensus exists regarding the generation interval length, and a wide variety of interval lengths have been used in recent studies. This makes comparison between studies difficult, and questions the accuracy of divergence date estimations. Recent genealogy-based research suggests that the male generation interval is substantially longer than the female interval, and that both are greater than the values commonly used in genetics studies. This study evaluates each of these hypotheses in a broader cross-cultural context, using data from both nation states and recent hunter-gatherer societies. Both hypotheses are supported by this study; therefore, revised estimates of male, female, and overall human generation interval lengths are proposed. The nearly universal, cross-cultural nature of the evidence justifies using these proposed estimates in Y-chromosomal, mitochondrial, and autosomal DNA-based population divergence studies.

Performance calculations for 200-1000 MWe MHD/steam power plants

NASA Technical Reports Server (NTRS)

Staiger, P. J.

1981-01-01

The effects of MHD generator length, level of oxygen enrichment, and oxygen production power on the performance of MHD/steam power plants ranging from 200 to 1000 MW in electrical output are investigated. The plants considered use oxygen enriched combustion air preheated to 1100 F. Both plants in which the MHD generator is cooled with low temperature and pressure boiler feedwater and plants in which the generator is cooled with high temperature and pressure boiler feedwater are considered. For plants using low temperature boiler feedwater for generator cooling the maximum thermodynamic efficiency is obtained with shorter generators and a lower level of oxygen enrichment compared to plants using high temperature boiler feedwater for generator cooling. The generator length at which the maximum plant efficiency occurs increases with power plant size for plants with a generator cooled by low temperature feedwater. Also shown is the relationship of the magnet stored energy requirement of the generator length and the power plant performance. Possible cost/performance tradeoffs between magnet cost and plant performance are indicated.

Hydroxyurea enhances SMN2 gene expression through nitric oxide release.

PubMed

Xu, Cheng; Chen, Xin; Grzeschik, Susanna M; Ganta, Madhuri; Wang, Ching H

2011-02-01

Small molecules that increase full-length survivor motor neuron (SMN) gene transcript are promising therapeutic candidates for spinal muscular atrophy (SMA). Hydroxyurea (HU) has recently been shown to increase full-length SMN transcript in cultured lymphocytes from patients with SMA. We investigate the mechanism by which HU enhances full-length SMN2 gene expression in SMA lymphocytes. Nitric oxide (NO) is a major intracellular metabolite of HU. We test whether NO donors can themselves enhance full-length SMN2 expression. Eighteen cell lines (five type I, five type II, six type III SMA, and two non-SMA controls) were treated with or without NO donors for 48 h. SMA cells treated with HU and three NO donors: two long-acting donors, Deta-NONOate and S-nitrosoglutathione, and one short-acting donor, 3-ethyl-3-(ethylaminoethyl)-1-hydroxy-2-oxo-1-triazene, resulted in significant increase in full-length SMN2 mRNA. These effects were abolished by co-treatment with an NO scavenger 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide. One short-acting NO donor, S-nitroso-N-acetyl-DL-penicillamine, failed to show significant effect on full-length SMN2 expression, possibly due to high degree of cytotoxicity. These results were observed using both densitometry and quantitative PCR methods. We conclude that HU enhances SMN2 expression through the release of NO. NO donors may themselves be considered as new therapeutic candidates for SMA.

Bisphenol A (BPA) binding on full-length architectures of estrogen receptor.

PubMed

Liu, Yaquan; Qu, Kaili; Hai, Ying; Zhao, Chunyan

2018-08-01

Previous research has shown that the major toxicity mechanism for many environment chemicals is binding with estrogen receptor (ER) and blocking endogenous estrogen access, including bisphenol A (BPA). However, the molecular level understanding the global consequence of BPA binding on the full-length architectures of ER is largely unknown, which is a necessary stage to evaluate estrogen-like toxicity of BPA. In the present work, the consequence of BPA on full-length architectures of ER was firstly modeled based on molecular dynamics, focusing on the cross communication between multi-domains including ligand binding domain (LBD) and DNA binding domain (DBD). The study proved consequence of BPA upon full-length ER structure was dependent on long-range communications between multiple protein domains. The allosteric effects occurring in LBD units could alter dimerization formation through a crucial change in residue-residue connections, which resulted in relaxation of DBD. It indicated BPA could present consequence on the full-size receptor, not only on the separate domains, but also on the cross communication among LBD, DBD, and DNA molecules. It might provide detailed insight into the knowledge about the structural characteristics of ER and its role in gene regulation, which eventually helped us evaluate the estrogen-like toxicity upon BPA binding with full-length ER. © 2018 Wiley Periodicals, Inc.

Application of linker technique to trap transiently interacting protein complexes for structural studies

PubMed Central

Reddy Chichili, Vishnu Priyanka; Kumar, Veerendra; Sivaraman, J.

2016-01-01

Protein-protein interactions are key events controlling several biological processes. We have developed and employed a method to trap transiently interacting protein complexes for structural studies using glycine-rich linkers to fuse interacting partners, one of which is unstructured. Initial steps involve isothermal titration calorimetry to identify the minimum binding region of the unstructured protein in its interaction with its stable binding partner. This is followed by computational analysis to identify the approximate site of the interaction and to design an appropriate linker length. Subsequently, fused constructs are generated and characterized using size exclusion chromatography and dynamic light scattering experiments. The structure of the chimeric protein is then solved by crystallization, and validated both in vitro and in vivo by substituting key interacting residues of the full length, unlinked proteins with alanine. This protocol offers the opportunity to study crucial and currently unattainable transient protein interactions involved in various biological processes. PMID:26985443

Feeding-Related Traits Are Affected by Dosage of the foraging Gene in Drosophila melanogaster

PubMed Central

Allen, Aaron M.; Anreiter, Ina; Neville, Megan C.; Sokolowski, Marla B.

2017-01-01

Nutrient acquisition and energy storage are critical parts of achieving metabolic homeostasis. The foraging gene in Drosophila melanogaster has previously been implicated in multiple feeding-related and metabolic traits. Before foraging’s functions can be further dissected, we need a precise genetic null mutant to definitively map its amorphic phenotypes. We used homologous recombination to precisely delete foraging, generating the for0 null allele, and used recombineering to reintegrate a full copy of the gene, generating the {forBAC} rescue allele. We show that a total loss of foraging expression in larvae results in reduced larval path length and food intake behavior, while conversely showing an increase in triglyceride levels. Furthermore, varying foraging gene dosage demonstrates a linear dose-response on these phenotypes in relation to foraging gene expression levels. These experiments have unequivocally proven a causal, dose-dependent relationship between the foraging gene and its pleiotropic influence on these feeding-related traits. Our analysis of foraging’s transcription start sites, termination sites, and splicing patterns using rapid amplification of cDNA ends (RACE) and full-length cDNA sequencing, revealed four independent promoters, pr1–4, that produce 21 transcripts with nine distinct open reading frames (ORFs). The use of alternative promoters and alternative splicing at the foraging locus creates diversity and flexibility in the regulation of gene expression, and ultimately function. Future studies will exploit these genetic tools to precisely dissect the isoform- and tissue-specific requirements of foraging’s functions and shed light on the genetic control of feeding-related traits involved in energy homeostasis. PMID:28007892

Filling reference gaps via assembling DNA barcodes using high-throughput sequencing—moving toward barcoding the world

PubMed Central

Zhou, Chengran

2017-01-01

Abstract Over the past decade, biodiversity researchers have dedicated tremendous efforts to constructing DNA reference barcodes for rapid species registration and identification. Although analytical cost for standard DNA barcoding has been significantly reduced since early 2000, further dramatic reduction in barcoding costs is unlikely because Sanger sequencing is approaching its limits in throughput and chemistry cost. Constraints in barcoding cost not only led to unbalanced barcoding efforts around the globe, but also prevented high-throughput sequencing (HTS)–based taxonomic identification from applying binomial species names, which provide crucial linkages to biological knowledge. We developed an Illumina-based pipeline, HIFI-Barcode, to produce full-length Cytochrome c oxidase subunit I (COI) barcodes from pooled polymerase chain reaction amplicons generated by individual specimens. The new pipeline generated accurate barcode sequences that were comparable to Sanger standards, even for different haplotypes of the same species that were only a few nucleotides different from each other. Additionally, the new pipeline was much more sensitive in recovering amplicons at low quantity. The HIFI-Barcode pipeline successfully recovered barcodes from more than 78% of the polymerase chain reactions that didn’t show clear bands on the electrophoresis gel. Moreover, sequencing results based on the single molecular sequencing platform Pacbio confirmed the accuracy of the HIFI-Barcode results. Altogether, the new pipeline can provide an improved solution to produce full-length reference barcodes at about one-tenth of the current cost, enabling construction of comprehensive barcode libraries for local fauna, leading to a feasible direction for DNA barcoding global biomes. PMID:29077841

Filling reference gaps via assembling DNA barcodes using high-throughput sequencing-moving toward barcoding the world.

PubMed

Liu, Shanlin; Yang, Chentao; Zhou, Chengran; Zhou, Xin

2017-12-01

Over the past decade, biodiversity researchers have dedicated tremendous efforts to constructing DNA reference barcodes for rapid species registration and identification. Although analytical cost for standard DNA barcoding has been significantly reduced since early 2000, further dramatic reduction in barcoding costs is unlikely because Sanger sequencing is approaching its limits in throughput and chemistry cost. Constraints in barcoding cost not only led to unbalanced barcoding efforts around the globe, but also prevented high-throughput sequencing (HTS)-based taxonomic identification from applying binomial species names, which provide crucial linkages to biological knowledge. We developed an Illumina-based pipeline, HIFI-Barcode, to produce full-length Cytochrome c oxidase subunit I (COI) barcodes from pooled polymerase chain reaction amplicons generated by individual specimens. The new pipeline generated accurate barcode sequences that were comparable to Sanger standards, even for different haplotypes of the same species that were only a few nucleotides different from each other. Additionally, the new pipeline was much more sensitive in recovering amplicons at low quantity. The HIFI-Barcode pipeline successfully recovered barcodes from more than 78% of the polymerase chain reactions that didn't show clear bands on the electrophoresis gel. Moreover, sequencing results based on the single molecular sequencing platform Pacbio confirmed the accuracy of the HIFI-Barcode results. Altogether, the new pipeline can provide an improved solution to produce full-length reference barcodes at about one-tenth of the current cost, enabling construction of comprehensive barcode libraries for local fauna, leading to a feasible direction for DNA barcoding global biomes. © The Authors 2017. Published by Oxford University Press.

Single-molecule, full-length transcript sequencing provides insight into the extreme metabolism of the ruby-throated hummingbird Archilochus colubris.

PubMed

Workman, Rachael E; Myrka, Alexander M; Wong, G William; Tseng, Elizabeth; Welch, Kenneth C; Timp, Winston

2018-03-01

Hummingbirds oxidize ingested nectar sugars directly to fuel foraging but cannot sustain this fuel use during fasting periods, such as during the night or during long-distance migratory flights. Instead, fasting hummingbirds switch to oxidizing stored lipids that are derived from ingested sugars. The hummingbird liver plays a key role in moderating energy homeostasis and this remarkable capacity for fuel switching. Additionally, liver is the principle location of de novo lipogenesis, which can occur at exceptionally high rates, such as during premigratory fattening. Yet understanding how this tissue and whole organism moderates energy turnover is hampered by a lack of information regarding how relevant enzymes differ in sequence, expression, and regulation. We generated a de novo transcriptome of the hummingbird liver using PacBio full-length cDNA sequencing (Iso-Seq), yielding 8.6Gb of sequencing data, or 2.6M reads from 4 different size fractions. We analyzed data using the SMRTAnalysis v3.1 Iso-Seq pipeline, then clustered isoforms into gene families to generate de novo gene contigs using Cogent. We performed orthology analysis to identify closely related sequences between our transcriptome and other avian and human gene sets. Finally, we closely examined homology of critical lipid metabolism genes between our transcriptome data and avian and human genomes. We confirmed high levels of sequence divergence within hummingbird lipogenic enzymes, suggesting a high probability of adaptive divergent function in the hepatic lipogenic pathways. Our results leverage cutting-edge technology and a novel bioinformatics pipeline to provide a first direct look at the transcriptome of this incredible organism.

RNA Polymerase II Elongation Control

PubMed Central

Zhou, Qiang; Li, Tiandao; Price, David H.

2014-01-01

Regulation of the elongation phase of transcription by RNA Polymerase II (Pol II) is utilized extensively to generate the pattern of mRNAs needed to specify cell types and to respond to environmental changes. After Pol II initiates, negative elongation factors cause it to pause in a promoter proximal position. These polymerases are poised to respond to the positive transcription elongation factor, P-TEFb, and then enter productive elongation only under the appropriate set of signals to generate full length properly processed mRNAs. Recent global analyses of Pol II and elongation factors, mechanisms that regulate P-TEFb involving the 7SK snRNP, factors that control both the negative and positive elongation properties of Pol II and the mRNA processing events that are coupled with elongation are discussed. PMID:22404626

Epistasis and inheritance of plant habit and fruit quality traits in ornamental pepper (Capsicum annuum L.).

PubMed

Santos, R M C; do Rêgo, E R; Borém, A; Nascimento, M F; Nascimento, N F F; Finger, F L; Rêgo, M M

2014-10-31

Two accessions of ornamental pepper Capsicum annuum L., differing in most of the characters studied, were crossed, resulting in the F1 generation, and the F2 generation was obtained through self-fertilization of the F1 generation. The backcross generations RC1 and RC2 were obtained through crossing between F1 and the parents P1 and P2, respectively. Morpho-agronomic characterization was performed based on the 19 quantitative descriptors of Capsicum. The data obtained were subjected to generation analysis, in which the means and additive variance (σa(2)), variance due to dominance deviation (σd(2)), phenotypic variance (σf(2)), genetic variance (σg(2)) and environmental variance (σm(2)) were calculated. For the full model, we estimated the mean effects of all possible homozygotes, additives, dominants, and epistatics: additive-additive, additive-dominant, and dominant-dominant. For the additive-dominant model, we estimated the additive effects, dominant effects and mean effects of possible homozygotes. The character fruit dry matter had the lowest value for broad sense heritability (0.42), and the highest values were found for fresh matter and fruit weight, 0.91 and 0.92, respectively. The lowest value for narrow sense heritability was for the minor fruit diameter character (0.33), and the highest values were found for seed yield per fruit and fresh matter, 0.87 and 0.84, respectively. The additive-dominant model explained only the variation found in plant height, canopy width, stem length, corolla diameter, leaf width, and pedicel length, but in the other characters, the epistatic effects showed significant values.

Estimating inbreeding in large, semi-isolated populations: effects of varying generation lengths and of migration.

PubMed

Pattison, J E

2007-01-01

The purpose of the study reported here was to investigate two important assumptions used in a recently reported new method of estimating inbreeding in large, relatively isolated populations over historic times. The method, based on modeling the genealogical "paradox," produces values of Pearl's coefficients, Z, a measure of inbreeding or genealogical coalescence, as a function of time. In this study, the effects on inbreeding of two important assumptions made in earlier studies, namely those of using a constant generation length and of ignoring migration, have been investigated for the population of Britain. First, by relating the median age of women at childbirth to the development level of various societies, the variation of the generation lengths for different periods in historic Britain were estimated. Values of Z for two types of varying generation lengths were then calculated and compared with the case of constant generation length. Second, the population curve for Britain used in earlier studies was modified to obtain the subpopulation at any time during the past two millennia that was descended from the pre-Roman British Celts. Values of Z for the case with migration were then calculated and compared with the case for no migration. It is shown that these two assumptions may be taken into account if and when required. Both the effect of a varying generation length and the effect of migration on Z were found to be 20-40%, when no known value of inbreeding was used, and 2-5%, when a known value of inbreeding was used.

A Method for Estimating Noise from Full-Scale Distributed Exhaust Nozzles

NASA Technical Reports Server (NTRS)

Kinzie, Kevin W.; Schein, David B.

2004-01-01

A method to estimate the full-scale noise suppression from a scale model distributed exhaust nozzle (DEN) is presented. For a conventional scale model exhaust nozzle, Strouhal number scaling using a scale factor related to the nozzle exit area is typically applied that shifts model scale frequency in proportion to the geometric scale factor. However, model scale DEN designs have two inherent length scales. One is associated with the mini-nozzles, whose size do not change in going from model scale to full scale. The other is associated with the overall nozzle exit area which is much smaller than full size. Consequently, lower frequency energy that is generated by the coalesced jet plume should scale to lower frequency, but higher frequency energy generated by individual mini-jets does not shift frequency. In addition, jet-jet acoustic shielding by the array of mini-nozzles is a significant noise reduction effect that may change with DEN model size. A technique has been developed to scale laboratory model spectral data based on the premise that high and low frequency content must be treated differently during the scaling process. The model-scale distributed exhaust spectra are divided into low and high frequency regions that are then adjusted to full scale separately based on different physics-based scaling laws. The regions are then recombined to create an estimate of the full-scale acoustic spectra. These spectra can then be converted to perceived noise levels (PNL). The paper presents the details of this methodology and provides an example of the estimated noise suppression by a distributed exhaust nozzle compared to a round conic nozzle.

[Construction of dengue virus-specific full-length fully human antibody libraries by mammalian display technology].

PubMed

Wen, Yangming; Lan, Kaijian; Wang, Junjie; Yu, Jingyi; Qu, Yarong; Zhao, Wei; Zhang, Fuchun; Tan, Wanlong; Cao, Hong; Zhou, Chen

2013-06-01

To construct dengue virus-specific full-length fully human antibody libraries using mammalian cell surface display technique. Total RNA was extracted from peripheral blood mononuclear cells (PBMCs) from convalescent patients with dengue fever. The reservoirs of the light chain and heavy chain variable regions (LCκ and VH) of the antibody genes were amplified by RT-PCR and inserted into the vector pDGB-HC-TM separately to construct the light chain and heavy chain libraries. The library DNAs were transfected into CHO cells and the expression of full-length fully human antibodies on the surface of CHO cells was analyzed by flow cytometry. Using 1.2 µg of the total RNA isolated from the PBMCs as the template, the LCκ and VH were amplified and the full-length fully human antibody mammalian display libraries were constructed. The kappa light chain gene library had a size of 1.45×10(4) and the heavy chain gene library had a size of 1.8×10(5). Sequence analysis showed that 8 out of the 10 light chain clones and 7 out of the 10 heavy chain clones randomly picked up from the constructed libraries contained correct open reading frames. FACS analysis demonstrated that all the 15 clones with correct open reading frames expressed full-length antibodies, which could be detected on CHO cell surfaces. After co-transfection of the heavy chain and light chain gene libraries into CHO cells, the expression of full-length antibodies on CHO cell surfaces could be detected by FACS analysis with an expressible diversity of the antibody library reaching 1.46×10(9) [(1.45×10(4)×80%)×(1.8×10(5)×70%)]. Using 1.2 µg of total RNA as template, the LCκ and VH full-length fully human antibody libraries against dengue virus have been successfully constructed with an expressible diversity of 10(9).

Length oscillation mimicking periodic individual deep inspirations during tidal breathing attenuates force recovery and adaptation in airway smooth muscle.

PubMed

Raqeeb, Abdul; Solomon, Dennis; Paré, Peter D; Seow, Chun Y

2010-11-01

Airway smooth muscle (ASM) is able to generate maximal force under static conditions, and this isometric force can be maintained over a large length range due to length adaptation. The increased force at short muscle length could lead to excessive narrowing of the airways. Prolonged exposure of ASM to submaximal stimuli also increases the muscle's ability to generate force in a process called force adaptation. To date, the effects of length and force adaptation have only been demonstrated under static conditions. In the mechanically dynamic environment of the lung, ASM is constantly subjected to periodic stretches by the parenchyma due to tidal breathing and deep inspiration. It is not known whether force recovery due to muscle adaptation to a static environment could occur in a dynamic environment. In this study the effect of length oscillation mimicking tidal breathing and deep inspiration was examined. Force recovery after a length change was attenuated in the presence of length oscillation, except at very short lengths. Force adaptation was abolished by length oscillation. We conclude that in a healthy lung (with intact airway-parenchymal tethering) where airways are not allowed to narrow excessively, large stretches (associated with deep inspiration) may prevent the ability of the muscle to generate maximal force that would occur under static conditions irrespective of changes in mean length; mechanical perturbation on ASM due to tidal breathing and deep inspiration, therefore, is the first line of defense against excessive bronchoconstriction that may result from static length and force adaptation.

Assessing self-care and social function using a computer adaptive testing version of the pediatric evaluation of disability inventory.

PubMed

Coster, Wendy J; Haley, Stephen M; Ni, Pengsheng; Dumas, Helene M; Fragala-Pinkham, Maria A

2008-04-01

To examine score agreement, validity, precision, and response burden of a prototype computer adaptive testing (CAT) version of the self-care and social function scales of the Pediatric Evaluation of Disability Inventory compared with the full-length version of these scales. Computer simulation analysis of cross-sectional and longitudinal retrospective data; cross-sectional prospective study. Pediatric rehabilitation hospital, including inpatient acute rehabilitation, day school program, outpatient clinics; community-based day care, preschool, and children's homes. Children with disabilities (n=469) and 412 children with no disabilities (analytic sample); 38 children with disabilities and 35 children without disabilities (cross-validation sample). Not applicable. Summary scores from prototype CAT applications of each scale using 15-, 10-, and 5-item stopping rules; scores from the full-length self-care and social function scales; time (in seconds) to complete assessments and respondent ratings of burden. Scores from both computer simulations and field administration of the prototype CATs were highly consistent with scores from full-length administration (r range, .94-.99). Using computer simulation of retrospective data, discriminant validity, and sensitivity to change of the CATs closely approximated that of the full-length scales, especially when the 15- and 10-item stopping rules were applied. In the cross-validation study the time to administer both CATs was 4 minutes, compared with over 16 minutes to complete the full-length scales. Self-care and social function score estimates from CAT administration are highly comparable with those obtained from full-length scale administration, with small losses in validity and precision and substantial decreases in administration time.

Importance of Viral Sequence Length and Number of Variable and Informative Sites in Analysis of HIV Clustering.

PubMed

Novitsky, Vlad; Moyo, Sikhulile; Lei, Quanhong; DeGruttola, Victor; Essex, M

2015-05-01

To improve the methodology of HIV cluster analysis, we addressed how analysis of HIV clustering is associated with parameters that can affect the outcome of viral clustering. The extent of HIV clustering and tree certainty was compared between 401 HIV-1C near full-length genome sequences and subgenomic regions retrieved from the LANL HIV Database. Sliding window analysis was based on 99 windows of 1,000 bp and 45 windows of 2,000 bp. Potential associations between the extent of HIV clustering and sequence length and the number of variable and informative sites were evaluated. The near full-length genome HIV sequences showed the highest extent of HIV clustering and the highest tree certainty. At the bootstrap threshold of 0.80 in maximum likelihood (ML) analysis, 58.9% of near full-length HIV-1C sequences but only 15.5% of partial pol sequences (ViroSeq) were found in clusters. Among HIV-1 structural genes, pol showed the highest extent of clustering (38.9% at a bootstrap threshold of 0.80), although it was significantly lower than in the near full-length genome sequences. The extent of HIV clustering was significantly higher for sliding windows of 2,000 bp than 1,000 bp. We found a strong association between the sequence length and proportion of HIV sequences in clusters, and a moderate association between the number of variable and informative sites and the proportion of HIV sequences in clusters. In HIV cluster analysis, the extent of detectable HIV clustering is directly associated with the length of viral sequences used, as well as the number of variable and informative sites. Near full-length genome sequences could provide the most informative HIV cluster analysis. Selected subgenomic regions with a high extent of HIV clustering and high tree certainty could also be considered as a second choice.

Importance of Viral Sequence Length and Number of Variable and Informative Sites in Analysis of HIV Clustering

PubMed Central

Novitsky, Vlad; Moyo, Sikhulile; Lei, Quanhong; DeGruttola, Victor

2015-01-01

Abstract To improve the methodology of HIV cluster analysis, we addressed how analysis of HIV clustering is associated with parameters that can affect the outcome of viral clustering. The extent of HIV clustering and tree certainty was compared between 401 HIV-1C near full-length genome sequences and subgenomic regions retrieved from the LANL HIV Database. Sliding window analysis was based on 99 windows of 1,000 bp and 45 windows of 2,000 bp. Potential associations between the extent of HIV clustering and sequence length and the number of variable and informative sites were evaluated. The near full-length genome HIV sequences showed the highest extent of HIV clustering and the highest tree certainty. At the bootstrap threshold of 0.80 in maximum likelihood (ML) analysis, 58.9% of near full-length HIV-1C sequences but only 15.5% of partial pol sequences (ViroSeq) were found in clusters. Among HIV-1 structural genes, pol showed the highest extent of clustering (38.9% at a bootstrap threshold of 0.80), although it was significantly lower than in the near full-length genome sequences. The extent of HIV clustering was significantly higher for sliding windows of 2,000 bp than 1,000 bp. We found a strong association between the sequence length and proportion of HIV sequences in clusters, and a moderate association between the number of variable and informative sites and the proportion of HIV sequences in clusters. In HIV cluster analysis, the extent of detectable HIV clustering is directly associated with the length of viral sequences used, as well as the number of variable and informative sites. Near full-length genome sequences could provide the most informative HIV cluster analysis. Selected subgenomic regions with a high extent of HIV clustering and high tree certainty could also be considered as a second choice. PMID:25560745

Analysis of a Rocket Based Combined Cycle Engine during Rocket Only Operation

NASA Technical Reports Server (NTRS)

Smith, T. D.; Steffen, C. J., Jr.; Yungster, S.; Keller, D. J.

1998-01-01

The all rocket mode of operation is a critical factor in the overall performance of a rocket based combined cycle (RBCC) vehicle. However, outside of performing experiments or a full three dimensional analysis, there are no first order parametric models to estimate performance. As a result, an axisymmetric RBCC engine was used to analytically determine specific impulse efficiency values based upon both full flow and gas generator configurations. Design of experiments methodology was used to construct a test matrix and statistical regression analysis was used to build parametric models. The main parameters investigated in this study were: rocket chamber pressure, rocket exit area ratio, percent of injected secondary flow, mixer-ejector inlet area, mixer-ejector area ratio, and mixer-ejector length-to-inject diameter ratio. A perfect gas computational fluid dynamics analysis was performed to obtain values of vacuum specific impulse. Statistical regression analysis was performed based on both full flow and gas generator engine cycles. Results were also found to be dependent upon the entire cycle assumptions. The statistical regression analysis determined that there were five significant linear effects, six interactions, and one second-order effect. Two parametric models were created to provide performance assessments of an RBCC engine in the all rocket mode of operation.

SU-F-J-48: Effect of Scan Length On Magnitude of Imaging Dose in KV CBCT

DOE Office of Scientific and Technical Information (OSTI.GOV)

Deshpande, S; Naidu, S; Sutar, A

Purpose: To study effect of scan length on magnitude of imaging dose deposition in Varian kV CBCT for head & neck and pelvis CBCT. Methods: To study effect of scan length we measured imaging dose at depth of 8 cm for head and neck Cone Beam Computed Tomography (CBCT) acquisition ( X ray beam energy is used 100kV and 200 degree of gantry rotation) and at 16 cm depth for pelvis CBCT acquisition ( X ray beam energy used is 125 kV and 360 degree of gantry rotation) in specially designed phantom. We used farmer chamber which was calibrated inmore » kV X ray range for measurements .Dose was measured with default field size, and reducing field size along y direction to 10 cm and 5 cm. Results: As the energy of the beam decreases the scattered radiation increases and this contributes significantly to the dose deposited in the patient. By reducing the scan length to 10 Cm from default 20.6 cm we found a dose reduction of 14% for head and neck CBCT protocol and a reduction of 26% for pelvis CBCT protocol. Similarly for a scan length of 5cm compared to default the dose reduction in head and neck CBCT protocol is 36% while in the pelvis CBCT protocol the dose reduction is 50%. Conclusion: By limiting the scan length we can control the scatter radiation generated and hence the dose to the patient. However the variation in dose reduction for same length used in two protocols is because of the scan geometry. The pelvis CBCT protocol uses a full rotation and head and neck CBCT protocol uses partial rotation.« less

Tapered fluorotellurite microstructured fibers for broadband supercontinuum generation.

PubMed

Wang, Fang; Wang, Kangkang; Yao, Chuanfei; Jia, Zhixu; Wang, Shunbin; Wu, Changfeng; Qin, Guanshi; Ohishi, Yasutake; Qin, Weiping

2016-02-01

Fluorotellurite microstructured fibers (MFs) based on TeO2-BaF2-Y2O3 glasses are fabricated by using a rod-in-tube method. Tapered fluorotellurite MFs with varied transition region lengths are prepared by employing an elongation machine. By using a tapered fluorotellurite MF with a transition region length of ∼3.3  cm as the nonlinear medium and a 1560 nm femtosecond fiber laser as the pump source, broadband supercontinuum generation covering from 470 to 2770 nm is obtained. The effects of the transition region length of the tapered fluorotellurite MF on supercontinuum generation are also investigated. Our results show that tapered fluorotellurite MFs are promising nonlinear media for generating broadband supercontinuum light expanding from visible to mid-infrared spectral region.

Temperature-sensitive elastin-mimetic dendrimers: Effect of peptide length and dendrimer generation to temperature sensitivity.

PubMed

Kojima, Chie; Irie, Kotaro; Tada, Tomoko; Tanaka, Naoki

2014-06-01

Dendrimers are synthetic macromolecules with unique structure, which are a potential scaffold for peptides. Elastin is one of the main components of extracellular matrix and a temperature-sensitive biomacromolecule. Previously, Val-Pro-Gly-Val-Gly peptides have been conjugated to a dendrimer for designing an elastin-mimetic dendrimer. In this study, various elastin-mimetic dendrimers using different length peptides and different dendrimer generations were synthesized to control the temperature dependency. The elastin-mimetic dendrimers formed β-turn structure by heating, which was similar to the elastin-like peptides. The elastin-mimetic dendrimers exhibited an inverse phase transition, largely depending on the peptide length and slightly depending on the dendrimer generation. The elastin-mimetic dendrimers formed aggregates after the phase transition. The endothermal peak was observed in elastin-mimetic dendrimers with long peptides, but not with short ones. The peptide length and the dendrimer generation are important factors to tune the temperature dependency on the elastin-mimetic dendrimer. Copyright © 2013 Wiley Periodicals, Inc.

Protein Structure and Function Prediction Using I-TASSER

PubMed Central

Yang, Jianyi; Zhang, Yang

2016-01-01

I-TASSER is a hierarchical protocol for automated protein structure prediction and structure-based function annotation. Starting from the amino acid sequence of target proteins, I-TASSER first generates full-length atomic structural models from multiple threading alignments and iterative structural assembly simulations followed by atomic-level structure refinement. The biological functions of the protein, including ligand-binding sites, enzyme commission number, and gene ontology terms, are then inferred from known protein function databases based on sequence and structure profile comparisons. I-TASSER is freely available as both an on-line server and a stand-alone package. This unit describes how to use the I-TASSER protocol to generate structure and function prediction and how to interpret the prediction results, as well as alternative approaches for further improving the I-TASSER modeling quality for distant-homologous and multi-domain protein targets. PMID:26678386

Frequency-resolved optical gating system with a tellurium crystal for characterizing free-electron lasers in the wavelength range of 10-30 {mu}m

DOE Office of Scientific and Technical Information (OSTI.GOV)

Iijima, Hokuto; Nagai, Ryoji; Nishimori, Nobuyuki

2009-12-15

A second-harmonic generation frequency-resolved optical gating (SHG-FROG) system has been developed for the complete characterization of laser pulses in the wavelength range of 10-30 {mu}m. A tellurium crystal is used so that spectrally resolved autocorrelation signals with a good signal-to-noise ratio are obtained. Pulses (wavelength {approx}22 {mu}m) generated from a free-electron laser are measured by the SHG-FROG system. The SHG intensity profile and the spectrum obtained by FROG measurements are well consistent with those of independent measurements of the pulse length and spectrum. The pulse duration and spectral width determined from the FROG trace are 0.6 ps and 5.2 THzmore » at full width half maximum, respectively.« less

Beyond sequencing: optical mapping of DNA in the age of nanotechnology and nanoscopy.

PubMed

Levy-Sakin, Michal; Ebenstein, Yuval

2013-08-01

Next generation sequencing (NGS) is revolutionizing all fields of biological research but it fails to extract the full range of information associated with genetic material. Optical mapping of DNA grants access to genetic and epigenetic information on individual DNA molecules up to ∼1 Mbp in length. Fluorescent labeling of specific sequence motifs, epigenetic marks and other genomic information on individual DNA molecules generates a high content optical barcode along the DNA. By stretching the DNA to a linear configuration this barcode may be directly visualized by fluorescence microscopy. We discuss the advances of these methods in light of recent developments in nano-fabrication and super-resolution optical imaging (nanoscopy) and review the latest achievements of optical mapping in the context of genomic analysis. Copyright © 2013 Elsevier Ltd. All rights reserved.

Contribution of photocurrent mechanism and influence of plasma length in THz generation by two-color laser induced plasma

NASA Astrophysics Data System (ADS)

Alirezaee, Hajar; Sharifian, Mehdi

2018-04-01

The contribution of the photocurrent mechanism has been investigated in terahertz (THz) generation in laser-induced gas plasma. This study was done through comparing the THz amplitude for two phase differences (" separators="| φ = 0 , π / 2 ) between the fundamental pulse and its second harmonic. Our particle-in-cell simulation result showed that this mechanism played the most important role in the two-color regime with a low-frequency range. We also investigated the effect of the plasma channel length on generated THz amplitude, and it was found that THz amplitude would depend significantly on the plasma channel length.

Identification of full-length dentin matrix protein 1 in dentin and bone.

PubMed

Huang, Bingzhen; Maciejewska, Izabela; Sun, Yao; Peng, Tao; Qin, Disheng; Lu, Yongbo; Bonewald, Lynda; Butler, William T; Feng, Jian; Qin, Chunlin

2008-05-01

Dentin matrix protein 1 (DMP1) has been identified in the extracellular matrix (ECM) of dentin and bone as the processed NH(2)-terminal and COOH-terminal fragment. However, the full-length form of DMP1 has not been identified in these tissues. The focus of this investigation was to search for the intact full-length DMP1 in dentin and bone. We used two types of anti-DMP1 antibodies to identify DMP1: one type specifically recognizes the NH(2)-terminal region and the other type is only reactive to the COOH-terminal region of the DMP1 amino acid sequence. An approximately 105-kDa protein, extracted from the ECM of rat dentin and bone, was recognized by both types of antibodies; and the migration rate of this protein was identical to the recombinant mouse full-length DMP1 made in eukaryotic cells. We concluded that this approximately 105-kDa protein is the full-length form of DMP1, which is considerably less abundant than its processed fragments in the ECM of dentin and bone. We also detected the full-length form of DMP1 and its processed fragments in the extract of dental pulp/odontoblast complex dissected from rat teeth. In addition, immunofluorescence analysis showed that in MC3T3-E1 cells the NH(2)-terminal and COOH-terminal fragments of DMP1 are distributed differently. Our findings indicate that the majority of DMP1 must be cleaved within the cells that synthesize it and that minor amounts of uncleaved DMP1 molecules are secreted into the ECM of dentin and bone.

Assessing self-care and social function using a computer adaptive testing version of the Pediatric Evaluation of Disability Inventory Accepted for Publication, Archives of Physical Medicine and Rehabilitation

PubMed Central

Coster, Wendy J.; Haley, Stephen M.; Ni, Pengsheng; Dumas, Helene M.; Fragala-Pinkham, Maria A.

2009-01-01

Objective To examine score agreement, validity, precision, and response burden of a prototype computer adaptive testing (CAT) version of the Self-Care and Social Function scales of the Pediatric Evaluation of Disability Inventory (PEDI) compared to the full-length version of these scales. Design Computer simulation analysis of cross-sectional and longitudinal retrospective data; cross-sectional prospective study. Settings Pediatric rehabilitation hospital, including inpatient acute rehabilitation, day school program, outpatient clinics; community-based day care, preschool, and children’s homes. Participants Four hundred sixty-nine children with disabilities and 412 children with no disabilities (analytic sample); 38 children with disabilities and 35 children without disabilities (cross-validation sample). Interventions Not applicable. Main Outcome Measures Summary scores from prototype CAT applications of each scale using 15-, 10-, and 5-item stopping rules; scores from the full-length Self-Care and Social Function scales; time (in seconds) to complete assessments and respondent ratings of burden. Results Scores from both computer simulations and field administration of the prototype CATs were highly consistent with scores from full-length administration (all r’s between .94 and .99). Using computer simulation of retrospective data, discriminant validity and sensitivity to change of the CATs closely approximated that of the full-length scales, especially when the 15- and 10-item stopping rules were applied. In the cross-validation study the time to administer both CATs was 4 minutes, compared to over 16 minutes to complete the full-length scales. Conclusions Self-care and Social Function score estimates from CAT administration are highly comparable to those obtained from full-length scale administration, with small losses in validity and precision and substantial decreases in administration time. PMID:18373991

Pharmacological efficacy of anti-IL-1β scFv, Fab and full-length antibodies in treatment of rheumatoid arthritis.

PubMed

Qi, Jianying; Ye, Xianlong; Ren, Guiping; Kan, Fangming; Zhang, Yu; Guo, Mo; Zhang, Zhiyi; Li, Deshan

2014-02-01

Rheumatoid arthritis (RA) is a chronic autoimmune inflammatory disease that mainly causes the synovial joint inflammation and cartilage destruction. Interleukin-1β (IL-1β) is an important proinflammatory cytokine involved in the pathogenesis of RA. In this study, we constructed and expressed anti-IL-1β-full-length antibody in CHO-K1-SV, anti-IL-1β-Fab and anti-IL-1β-scFv in Rosetta. We compared the therapeutic efficacy of three anti-IL-1β antibodies for CIA mice. Mice with CIA were subcutaneously injected with humanized anti-IL-1β-scFv, anti-IL-1β-Fab or anti-IL-1β-full-length antibody. The effects of treatment were determined by arthritis severity score, autoreactive humoral, cellular immune responses, histological lesion and cytokines production. Compared with anti-IL-1β-scFv treatments, anti-IL-1β-Fab and anti-IL-1β-full-length antibody therapy resulted in more significant effect in alleviating the severity of arthritis by preventing bone damage and cartilage destruction, reducing humoral and cellular immune responses, and down-regulating the expression of IL-1β, IL-6, IL-2, IFN-γ, TNF-α and MMP-3 in inflammatory tissue. The therapeutic effects of anti-IL-1β-Fab and anti-IL-1β-full-length antibodies on CIA mice had no significant difference. However, production of anti-IL-1β-full-length antibody in eukaryotic system is, in general, time-consuming and more expensive than that of anti-IL-1β-Fab in prokaryotic systems. In conclusion, as a small molecule antibody, anti-IL-1β-Fab is an ideal candidate for RA therapy. Copyright © 2013 Elsevier Ltd. All rights reserved.

Pediatric Helicobacter pylori gastropathy demonstrates a unique pattern of gastric foveolar hyperplasia.

PubMed

Saghier, Sadaf; Schwarz, Steven M; Anderson, Virginia; Gupta, Raavi; Heidarian, Amin; Rabinowitz, Simon S

2018-04-25

Helicobacter pylori (Hp) are the most common agents causing gastric mucosal injury worldwide. Foveolar hyperplasia is a key component of the stomach's reaction to injury. This study examines histopathologic characteristics associated with Helicobacter pylori and with non- Helicobacter pylori-associated gastropathy in children and adolescents, and compares the prevalence of foveolar hyperplasia among these disease subgroups and normal control subjects. Eighty-one gastric antral and corpus biopsies from subjects 2-19 years of age were studied. Twenty-two subjects with Helicobacter pylori gastritis were compared to 23 with non-Helicobacter pylori gastropathy and to 36 controls (normal biopsies). Foveolar length, full mucosal thickness, and the foveolar length: full mucosal thickness ratio were derived by a morphometric technique previously developed to analyze adult gastric tissue. Compared to controls, Helicobacter pylori gastritis demonstrated significant increases in antral foveolar length (P < .0001), full mucosal thickness (P < .0001), as well as corpus foveolar length (P < .05) and corpus full mucosal thickness (P < .05). Non-Helicobacter pylori-associated gastropathy also was characterized by increased antral foveolar length (P < .0001) and full mucosal thickness (P < .001) but corresponding corpus measurements did not differ from controls. Antral foveolar length in non-Helicobacter pylori gastropathy was increased, when compared to Helicobacter pylori gastritis (P < .05), while corpus values were not. The non-Helicobacter pylori gastropathy group demonstrated increased antral foveolar length: full mucosal thickness ratios, compared with Helicobacter pylori gastritis (P < .001) and with normal controls (P < .0001). An objective, quantitative approach to measuring foveolar hyperplasia in adults was successfully applied to pediatric biopsies and yielded a richer characterization of gastric pathology in children. Foveolar hyperplasia appears to be a generalized phenomenon in the presence of pediatric Helicobacter pylori gastritis but is limited to the antrum in non-Helicobacter pylori gastropathy. © 2018 John Wiley & Sons Ltd.

Generation of a sub-diffraction hollow ring by shaping an azimuthally polarized wave

PubMed Central

Chen, Gang; Wu, Zhi-xiang; Yu, An-ping; Zhang, Zhi-hai; Wen, Zhong-quan; Zhang, Kun; Dai, Lu-ru; Jiang, Sen-lin; Li, Yu-yan; Chen, Li; Wang, Chang-tao; Luo, Xian-gang

2016-01-01

The generation of a sub-diffraction optical hollow ring is of great interest in various applications, such as optical microscopy, optical tweezers, and nanolithography. Azimuthally polarized light is a good candidate for creating an optical hollow ring structure. Various of methods have been proposed theoretically for generation of sub-wavelength hollow ring by focusing azimuthally polarized light, but without experimental demonstrations, especially for sub-diffraction focusing. Super-oscillation is a promising approach for shaping sub-diffraction optical focusing. In this paper, a planar sub-diffraction diffractive lens is proposed, which has an ultra-long focal length of 600 λ and small numerical aperture of 0.64. A sub-diffraction hollow ring is experimentally created by shaping an azimuthally polarized wave. The full-width-at-half-maximum of the hollow ring is 0.61 λ, which is smaller than the lens diffraction limit 0.78 λ, and the observed largest sidelobe intensity is only 10% of the peak intensity. PMID:27876885

Clonal expansion of genome-intact HIV-1 in functionally polarized Th1 CD4+ T cells

PubMed Central

Orlova-Fink, Nina; Einkauf, Kevin; Chowdhury, Fatema Z.; Sun, Xiaoming; Harrington, Sean; Kuo, Hsiao-Hsuan; Hua, Stephane; Chen, Hsiao-Rong; Ouyang, Zhengyu; Reddy, Kavidha; Dong, Krista; Ndung’u, Thumbi; Walker, Bruce D.; Rosenberg, Eric S.; Yu, Xu G.

2017-01-01

HIV-1 causes a chronic, incurable disease due to its persistence in CD4+ T cells that contain replication-competent provirus, but exhibit little or no active viral gene expression and effectively resist combination antiretroviral therapy (cART). These latently infected T cells represent an extremely small proportion of all circulating CD4+ T cells but possess a remarkable long-term stability and typically persist throughout life, for reasons that are not fully understood. Here we performed massive single-genome, near-full-length next-generation sequencing of HIV-1 DNA derived from unfractionated peripheral blood mononuclear cells, ex vivo-isolated CD4+ T cells, and subsets of functionally polarized memory CD4+ T cells. This approach identified multiple sets of independent, near-full-length proviral sequences from cART-treated individuals that were completely identical, consistent with clonal expansion of CD4+ T cells harboring intact HIV-1. Intact, near-full-genome HIV-1 DNA sequences that were derived from such clonally expanded CD4+ T cells constituted 62% of all analyzed genome-intact sequences in memory CD4 T cells, were preferentially observed in Th1-polarized cells, were longitudinally detected over a duration of up to 5 years, and were fully replication- and infection-competent. Together, these data suggest that clonal proliferation of Th1-polarized CD4+ T cells encoding for intact HIV-1 represents a driving force for stabilizing the pool of latently infected CD4+ T cells. PMID:28628034

Clonal expansion of genome-intact HIV-1 in functionally polarized Th1 CD4+ T cells.

PubMed

Lee, Guinevere Q; Orlova-Fink, Nina; Einkauf, Kevin; Chowdhury, Fatema Z; Sun, Xiaoming; Harrington, Sean; Kuo, Hsiao-Hsuan; Hua, Stephane; Chen, Hsiao-Rong; Ouyang, Zhengyu; Reddy, Kavidha; Dong, Krista; Ndung'u, Thumbi; Walker, Bruce D; Rosenberg, Eric S; Yu, Xu G; Lichterfeld, Mathias

2017-06-30

HIV-1 causes a chronic, incurable disease due to its persistence in CD4+ T cells that contain replication-competent provirus, but exhibit little or no active viral gene expression and effectively resist combination antiretroviral therapy (cART). These latently infected T cells represent an extremely small proportion of all circulating CD4+ T cells but possess a remarkable long-term stability and typically persist throughout life, for reasons that are not fully understood. Here we performed massive single-genome, near-full-length next-generation sequencing of HIV-1 DNA derived from unfractionated peripheral blood mononuclear cells, ex vivo-isolated CD4+ T cells, and subsets of functionally polarized memory CD4+ T cells. This approach identified multiple sets of independent, near-full-length proviral sequences from cART-treated individuals that were completely identical, consistent with clonal expansion of CD4+ T cells harboring intact HIV-1. Intact, near-full-genome HIV-1 DNA sequences that were derived from such clonally expanded CD4+ T cells constituted 62% of all analyzed genome-intact sequences in memory CD4 T cells, were preferentially observed in Th1-polarized cells, were longitudinally detected over a duration of up to 5 years, and were fully replication- and infection-competent. Together, these data suggest that clonal proliferation of Th1-polarized CD4+ T cells encoding for intact HIV-1 represents a driving force for stabilizing the pool of latently infected CD4+ T cells.

The influence of conjugation variables on the design and immunogenicity of a glycoconjugate vaccine against Salmonella Typhi

PubMed Central

Arcuri, M.; Di Benedetto, R.; Cunningham, A. F.; Saul, A.; MacLennan, C. A.

2017-01-01

In recent years there have been major efforts to develop glycoconjugate vaccines based on the Vi polysaccharide that will protect against Salmonella enterica Typhi infections, particularly typhoid fever, which remains a major public health concern in low-income countries. The design of glycoconjugate vaccines influences the immune responses they elicit. Here we systematically test the response in mice to Vi glycoconjugates that differ in Vi chain length (full-length and fragmented), carrier protein, conjugation chemistry, saccharide to protein ratio and size. We show that the length of Vi chains, but not the ultimate size of the conjugate, has an impact on the anti-Vi IgG immune response induced. Full-length Vi conjugates, independent of the carrier protein, induce peak IgG responses rapidly after just one immunization, and secondary immunization does not enhance the magnitude of these responses. Fragmented Vi linked to CRM197 and diphtheria toxoid, but not to tetanus toxoid, gives lower anti-Vi antibody responses after the first immunization than full-length Vi conjugates, but antibody titres are similar to those induced by full-length Vi conjugates following a second dose. The chemistry to conjugate Vi to the carrier protein, the linker used, and the saccharide to protein ratio do not significantly alter the response. We conclude that Vi length and carrier protein are the variables that influence the anti-Vi IgG response to immunization the most, while other parameters are of lesser importance. PMID:29287062

Identification of a new hepatitis B virus recombinant D2/D3 in the city of São Paulo, Brazil.

PubMed

Santana, Luiz Claudio; Mantovani, Nathalia Pena; Ferreira, Maira Cicero; Arnold, Rafael; Duro, Rodrigo Lopes Sanz; Ferreira, Paulo Roberto Abrão; Hunter, James Richard; Leal, Élcio; Diaz, Ricardo Sobhie; Komninakis, Shirley Vasconcelos

2017-02-01

Two hundred forty million people are chronically infected with hepatitis B virus (HBV) worldwide. The rise of globalization has facilitated the emergence of novel HBV recombinants and genotypes. We evaluated HBV genotypes and recombinants, mutations associated with resistance to antivirals (AVs), progression of hepatic illness, and inefficient hepatitis B vaccination responses in chronically infected individuals in the city of São Paulo, Brazil. Forty-five full-length and 24 partial-length sequences were obtained. The genotype distribution was as follows: A (66.7%), D (15.9%), F (11.6%) and C (4.3%). We describe a new recombinant (D2/D3), confirmed through next-generation sequencing (NGS) and reconstruction of the quasispecies sequences in silico. Primary resistance and major vaccine escape mutations were not found. We did, however, find mutations in the S region that might may be related to HBV antigenicity changes, as well as Pre-S deletions. The precore/core mutations A1762T + G1764A (40.9%) were found mostly in genotypes A and D, and G1896A (29.55%) was more frequent in genotype D than in genotype A. The genotypic distribution reflects the history of Brazilian immigration. This is the first description of recombination between genotypes D2 and D3 in Brazil. It is also the first confirmation through NGS and reconstruction of the quasispecies in silico. However, little is known about the response to treatment of recombinants. This demonstrates the need for molecular epidemiology studies involving the analysis of full-length HBV sequences.

Relative fascicle excursion effects on dynamic strength generation during gait in children with cerebral palsy.

PubMed

Martín Lorenzo, T; Lerma Lara, S; Martínez-Caballero, I; Rocon, E

2015-10-01

Evaluation of muscle structure gives us a better understanding of how muscles contribute to force generation which is significantly altered in children with cerebral palsy (CP). While most muscle structure parameters have shown to be significantly correlated to different expressions of strength development in children with CP and typically developing (TD) children, conflicting results are found for muscle fascicle length. Muscle fascicle length determines muscle excursion and velocity, and contrary to what might be expected, correlations of fascicle length to rate of force development have not been found for children with CP. The lack of correlation between muscle fascicle length and rate of force development in children with CP could be due, on the one hand, to the non-optimal joint position adopted for force generation on the isometric strength tests as compared to the position of TD children. On the other hand, the lack of correlation could be due to the erroneous assumption that muscle fascicle length is representative of sarcomere length. Thus, the relationship between muscle architecture parameters reflecting sarcomere length, such as relative fascicle excursions and dynamic power generation, should be assessed. Understanding of the underlying mechanisms of weakness in children with CP is key for individualized prescription and assessment of muscle-targeted interventions. Findings could imply the detection of children operating on the descending limb of the sarcomere length-tension curve, which in turn might be at greater risk of developing crouch gait. Furthermore, relative muscle fascicle excursions could be used as a predictive variable of outcomes related to crouch gait prevention treatments such as strength training. Copyright © 2015 Elsevier Ltd. All rights reserved.

Calorie restriction suppresses subgenomic mink cytopathic focus-forming murine leukemia virus transcription and frequency of genomic expression while impairing lymphoma formation.

PubMed Central

Shields, B A; Engelman, R W; Fukaura, Y; Good, R A; Day, N K

1991-01-01

Calorie restriction suppresses mammary proviral mRNA expression and protooncogene activation in breast tumor-prone C3H/Ou mice while inhibiting tumor formation. To determine whether the beneficial effects of chronic energy-intake restriction (CEIR) can be extended to an organ site of retrovirus-induced tumorigenesis where the dynamics of growth and sexual maturity are not paramount as they are in breast tissue, calorie restriction of 40% was imposed on thymic lymphoma-prone AKR mice when 4 weeks old. Recombination between various murine leukemia virus (MuLV) mRNAs, resulting in the generation of an 8.4-kilobase genomic-length transcript with mink cytopathic focus-forming (MCF) characteristics, is considered the proximal retroviral event in AKR lymphomagenesis. Thymic expression of subgenomic MCF MuLV mRNA was uniformly suppressed among 6- and 8-week-old CEIR mice (P less than 0.02). This suppression of MuLV transcription preceded a 25% reduction in the appearance of genomic-length MCF transcripts among CEIR mice and a 28% reduction in cumulative lymphoma mortality. The latency to median tumor incidence was extended greater than 3 months by calorie restriction, and median lifespan was extended approximately 50%. Survival curves for the full-fed and CEIR dietary cohorts were found to be significantly different (P less than 0.0001), with full-fed mice experiencing a 3 times greater risk of lymphoma mortality. These findings extend the known range of pathologic states influenced by CEIR in inbred mice and show that retroviral mechanisms involved in generation of lymphoid malignancy can be significantly impaired by calorie restriction. Images PMID:1763029

Modeling Treatment Response for Lamin A/C Related Dilated Cardiomyopathy in Human Induced Pluripotent Stem Cells.

PubMed

Lee, Yee-Ki; Lau, Yee-Man; Cai, Zhu-Jun; Lai, Wing-Hon; Wong, Lai-Yung; Tse, Hung-Fat; Ng, Kwong-Man; Siu, Chung-Wah

2017-07-28

Precision medicine is an emerging approach to disease treatment and prevention that takes into account individual variability in the environment, lifestyle, and genetic makeup of patients. Patient-specific human induced pluripotent stem cells hold promise to transform precision medicine into real-life clinical practice. Lamin A/C (LMNA)-related cardiomyopathy is the most common inherited cardiomyopathy in which a substantial proportion of mutations in the LMNA gene are of nonsense mutation. PTC124 induces translational read-through over the premature stop codon and restores production of the full-length proteins from the affected genes. In this study we generated human induced pluripotent stem cells-derived cardiomyocytes from patients who harbored different LMNA mutations (nonsense and frameshift) to evaluate the potential therapeutic effects of PTC124 in LMNA -related cardiomyopathy. We generated human induced pluripotent stem cells lines from 3 patients who carried distinctive mutations (R225X, Q354X, and T518fs) in the LMNA gene. The cardiomyocytes derived from these human induced pluripotent stem cells lines reproduced the pathophysiological hallmarks of LMNA -related cardiomyopathy. Interestingly, PTC124 treatment increased the production of full-length LMNA proteins in only the R225X mutant, not in other mutations. Functional evaluation experiments on the R225X mutant further demonstrated that PTC124 treatment not only reduced nuclear blebbing and electrical stress-induced apoptosis but also improved the excitation-contraction coupling of the affected cardiomyocytes. Using cardiomyocytes derived from human induced pluripotent stem cells carrying different LMNA mutations, we demonstrated that the effect of PTC124 is codon selective. A premature stop codon UGA appeared to be most responsive to PTC124 treatment. © 2017 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley.

Full-length amyloid precursor protein regulates lipoprotein metabolism and amyloid-β clearance in human astrocytes.

PubMed

Fong, Lauren K; Yang, Max M; Dos Santos Chaves, Rodrigo; Reyna, Sol M; Langness, Vanessa F; Woodruff, Grace; Roberts, Elizabeth A; Young, Jessica E; Goldstein, Lawrence S B

2018-06-01

Mounting evidence suggests that alterations in cholesterol homeostasis are involved in Alzheimer's disease (AD) pathogenesis. Amyloid precursor protein (APP) or multiple fragments generated by proteolytic processing of APP have previously been implicated in the regulation of cholesterol metabolism. However, the physiological function of APP in regulating lipoprotein homeostasis in astrocytes, which are responsible for de novo cholesterol biosynthesis and regulation in the brain, remains unclear. To address this, here we used CRISPR/Cas9 genome editing to generate isogenic APP-knockout (KO) human induced pluripotent stem cells (hiPSCs) and differentiated them into human astrocytes. We found that APP-KO astrocytes have reduced cholesterol and elevated levels of sterol regulatory element-binding protein (SREBP) target gene transcripts and proteins, which were both downstream consequences of reduced lipoprotein endocytosis. To elucidate which APP fragments regulate cholesterol homeostasis and examine whether familial AD mutations in APP affect lipoprotein metabolism, we analyzed an isogenic allelic series harboring the APP Swedish and APP V717F variants. Only astrocytes homozygous for the APP Swedish (APP Swe/Swe ) mutation, which had reduced full-length APP (FL APP) due to increased β-secretase cleavage, recapitulated the APP-KO phenotypes. Astrocytic internalization of amyloid-β (Aβ), another ligand for low-density lipoprotein (LDL) receptors, was also impaired in APP-KO and APP Swe/Swe astrocytes. Finally, impairing cleavage of FL APP through β-secretase inhibition in APP Swe/Swe astrocytes reversed the LDL and Aβ endocytosis defects. In conclusion, FL APP is involved in the endocytosis of LDL receptor ligands and required for proper cholesterol homeostasis and Aβ clearance in human astrocytes. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.

A DNA Vaccine against Yellow Fever Virus: Development and Evaluation

PubMed Central

Maciel, Milton; Cruz, Fábia da Silva Pereira; Cordeiro, Marli Tenório; da Motta, Márcia Archer; Cassemiro, Klécia Marília Soares de Melo; Maia, Rita de Cássia Carvalho; de Figueiredo, Regina Célia Bressan Queiroz; Galler, Ricardo; Freire, Marcos da Silva; August, Joseph Thomas; Marques, Ernesto T. A.; Dhalia, Rafael

2015-01-01

Attenuated yellow fever (YF) virus 17D/17DD vaccines are the only available protection from YF infection, which remains a significant source of morbidity and mortality in the tropical areas of the world. The attenuated YF virus vaccine, which is used worldwide, generates both long-lasting neutralizing antibodies and strong T-cell responses. However, on rare occasions, this vaccine has toxic side effects that can be fatal. This study presents the design of two non-viral DNA-based antigen formulations and the characterization of their expression and immunological properties. The two antigen formulations consist of DNA encoding the full-length envelope protein (p/YFE) or the full-length envelope protein fused to the lysosomal-associated membrane protein signal, LAMP-1 (pL/YFE), aimed at diverting antigen processing/presentation through the major histocompatibility complex II precursor compartments. The immune responses triggered by these formulations were evaluated in H2b and H2d backgrounds, corresponding to the C57Bl/6 and BALB/c mice strains, respectively. Both DNA constructs were able to induce very strong T-cell responses of similar magnitude against almost all epitopes that are also generated by the YF 17DD vaccine. The pL/YFE formulation performed best overall. In addition to the T-cell response, it was also able to stimulate high titers of anti-YF neutralizing antibodies comparable to the levels elicited by the 17DD vaccine. More importantly, the pL/YFE vaccine conferred 100% protection against the YF virus in intracerebrally challenged mice. These results indicate that pL/YFE DNA is an excellent vaccine candidate and should be considered for further developmental studies. PMID:25875109

A DNA vaccine against yellow fever virus: development and evaluation.

PubMed

Maciel, Milton; Cruz, Fábia da Silva Pereira; Cordeiro, Marli Tenório; da Motta, Márcia Archer; Cassemiro, Klécia Marília Soares de Melo; Maia, Rita de Cássia Carvalho; de Figueiredo, Regina Célia Bressan Queiroz; Galler, Ricardo; Freire, Marcos da Silva; August, Joseph Thomas; Marques, Ernesto T A; Dhalia, Rafael

2015-04-01

Attenuated yellow fever (YF) virus 17D/17DD vaccines are the only available protection from YF infection, which remains a significant source of morbidity and mortality in the tropical areas of the world. The attenuated YF virus vaccine, which is used worldwide, generates both long-lasting neutralizing antibodies and strong T-cell responses. However, on rare occasions, this vaccine has toxic side effects that can be fatal. This study presents the design of two non-viral DNA-based antigen formulations and the characterization of their expression and immunological properties. The two antigen formulations consist of DNA encoding the full-length envelope protein (p/YFE) or the full-length envelope protein fused to the lysosomal-associated membrane protein signal, LAMP-1 (pL/YFE), aimed at diverting antigen processing/presentation through the major histocompatibility complex II precursor compartments. The immune responses triggered by these formulations were evaluated in H2b and H2d backgrounds, corresponding to the C57Bl/6 and BALB/c mice strains, respectively. Both DNA constructs were able to induce very strong T-cell responses of similar magnitude against almost all epitopes that are also generated by the YF 17DD vaccine. The pL/YFE formulation performed best overall. In addition to the T-cell response, it was also able to stimulate high titers of anti-YF neutralizing antibodies comparable to the levels elicited by the 17DD vaccine. More importantly, the pL/YFE vaccine conferred 100% protection against the YF virus in intracerebrally challenged mice. These results indicate that pL/YFE DNA is an excellent vaccine candidate and should be considered for further developmental studies.

A novel humanized mouse model of Huntington disease for preclinical development of therapeutics targeting mutant huntingtin alleles.

PubMed

Southwell, Amber L; Skotte, Niels H; Villanueva, Erika B; Østergaard, Michael E; Gu, Xiaofeng; Kordasiewicz, Holly B; Kay, Chris; Cheung, Daphne; Xie, Yuanyun; Waltl, Sabine; Dal Cengio, Louisa; Findlay-Black, Hailey; Doty, Crystal N; Petoukhov, Eugenia; Iworima, Diepiriye; Slama, Ramy; Ooi, Jolene; Pouladi, Mahmoud A; Yang, X William; Swayze, Eric E; Seth, Punit P; Hayden, Michael R

2017-03-15

Huntington disease (HD) is a neurodegenerative disease caused by a mutation in the huntingtin (HTT) gene. HTT is a large protein, interacts with many partners and is involved in many cellular pathways, which are perturbed in HD. Therapies targeting HTT directly are likely to provide the most global benefit. Thus there is a need for preclinical models of HD recapitulating human HTT genetics. We previously generated a humanized mouse model of HD, Hu97/18, by intercrossing BACHD and YAC18 mice with knockout of the endogenous mouse HD homolog (Hdh). Hu97/18 mice recapitulate the genetics of HD, having two full-length, genomic human HTT transgenes heterozygous for the HD mutation and polymorphisms associated with HD in populations of Caucasian descent. We have now generated a companion model, Hu128/21, by intercrossing YAC128 and BAC21 mice on the Hdh-/- background. Hu128/21 mice have two full-length, genomic human HTT transgenes heterozygous for the HD mutation and polymorphisms associated with HD in populations of East Asian descent and in a minority of patients from other ethnic groups. Hu128/21 mice display a wide variety of HD-like phenotypes that are similar to YAC128 mice. Additionally, both transgenes in Hu128/21 mice match the human HTT exon 1 reference sequence. Conversely, the BACHD transgene carries a floxed, synthetic exon 1 sequence. Hu128/21 mice will be useful for investigations of human HTT that cannot be addressed in Hu97/18 mice, for developing therapies targeted to exon 1, and for preclinical screening of personalized HTT lowering therapies in HD patients of East Asian descent. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Single-molecule, full-length transcript sequencing provides insight into the extreme metabolism of the ruby-throated hummingbird Archilochus colubris

PubMed Central

Workman, Rachael E; Myrka, Alexander M; Wong, G William; Tseng, Elizabeth

2018-01-01

Abstract Background Hummingbirds oxidize ingested nectar sugars directly to fuel foraging but cannot sustain this fuel use during fasting periods, such as during the night or during long-distance migratory flights. Instead, fasting hummingbirds switch to oxidizing stored lipids that are derived from ingested sugars. The hummingbird liver plays a key role in moderating energy homeostasis and this remarkable capacity for fuel switching. Additionally, liver is the principle location of de novo lipogenesis, which can occur at exceptionally high rates, such as during premigratory fattening. Yet understanding how this tissue and whole organism moderates energy turnover is hampered by a lack of information regarding how relevant enzymes differ in sequence, expression, and regulation. Findings We generated a de novo transcriptome of the hummingbird liver using PacBio full-length cDNA sequencing (Iso-Seq), yielding 8.6Gb of sequencing data, or 2.6M reads from 4 different size fractions. We analyzed data using the SMRTAnalysis v3.1 Iso-Seq pipeline, then clustered isoforms into gene families to generate de novo gene contigs using Cogent. We performed orthology analysis to identify closely related sequences between our transcriptome and other avian and human gene sets. Finally, we closely examined homology of critical lipid metabolism genes between our transcriptome data and avian and human genomes. Conclusions We confirmed high levels of sequence divergence within hummingbird lipogenic enzymes, suggesting a high probability of adaptive divergent function in the hepatic lipogenic pathways. Our results leverage cutting-edge technology and a novel bioinformatics pipeline to provide a first direct look at the transcriptome of this incredible organism. PMID:29618047

Functional Human α7 Nicotinic Acetylcholine Receptor (nAChR) Generated from Escherichia coli.

PubMed

Tillman, Tommy S; Alvarez, Frances J D; Reinert, Nathan J; Liu, Chuang; Wang, Dawei; Xu, Yan; Xiao, Kunhong; Zhang, Peijun; Tang, Pei

2016-08-26

Human Cys-loop receptors are important therapeutic targets. High-resolution structures are essential for rational drug design, but only a few are available due to difficulties in obtaining sufficient quantities of protein suitable for structural studies. Although expression of proteins in E. coli offers advantages of high yield, low cost, and fast turnover, this approach has not been thoroughly explored for full-length human Cys-loop receptors because of the conventional wisdom that E. coli lacks the specific chaperones and post-translational modifications potentially required for expression of human Cys-loop receptors. Here we report the successful production of full-length wild type human α7nAChR from E. coli Chemically induced chaperones promote high expression levels of well-folded proteins. The choice of detergents, lipids, and ligands during purification determines the final protein quality. The purified α7nAChR not only forms pentamers as imaged by negative-stain electron microscopy, but also retains pharmacological characteristics of native α7nAChR, including binding to bungarotoxin and positive allosteric modulators specific to α7nAChR. Moreover, the purified α7nAChR injected into Xenopus oocytes can be activated by acetylcholine, choline, and nicotine, inhibited by the channel blockers QX-222 and phencyclidine, and potentiated by the α7nAChR specific modulators PNU-120596 and TQS. The successful generation of functional human α7nAChR from E. coli opens a new avenue for producing mammalian Cys-loop receptors to facilitate structure-based rational drug design. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

The Fate of Major Royal Jelly Proteins during Proteolytic Digestion in the Human Gastrointestinal Tract.

PubMed

Mureşan, Carmen I; Schierhorn, Angelika; Buttstedt, Anja

2018-04-25

Royal jelly (RJ) is a beehive product with a complex composition, major royal jelly proteins (MRJPs) being the most abundant proteins. Cell culture and animal studies suggest various biological activities for the full-length/native MRJPs. In the field of apitherapy, it is assumed that MRJPs can positively affect human health. However, whenever RJ is administered orally, the availability for assimilation in the gastrointestinal tract is a prerequisite for MRJPs to have any effect on humans. We here show that MRJPs vary in resistance to pepsin digestion with MRJP2 being most stable and still present as full-length protein after 24 h of digestion. In the intestinal phase, using trypsin and chymotrypsin, MRJPs are rapidly digested with MRJP2 again showing longest stability (40 min), suggesting that MRJPs can reach the small intestine as full-length proteins but then have to be resorbed quickly if full-length proteins are to fulfill any biological activity.

Identification of full-length proviral DNA of porcine endogenous retrovirus from Chinese Wuzhishan miniature pigs inbred.

PubMed

Ma, Yuyuan; Lv, Maomin; Xu, Shu; Wu, Jianmin; Tian, Kegong; Zhang, Jingang

2010-07-01

Existence of porcine endogenous retrovirus (PERV) hinders pigs to be used in clinical xenotransplantation to alleviate the shortage of human transplants. Chinese miniature pigs are potential organ donors for xenotransplantation in China. However, so far, an adequate level of information on the molecular characteristics of PERV from Chinese miniature pigs has not been available. We described here the cloning and characterization of full-length proviral DNA of PERV from Chinese Wuzhishan miniature pigs inbred (WZSP). Full-length nucleotide sequences of PERV-WZSP and other PERVs were aligned and phylogenetic tree was constructed from deduced amino-acid sequences of env. The results demonstrated that the full-length proviral DNA of PERV-WZSP belongs to gammaretrovirus and shares high similarity with other PERVs. Sequence analysis also suggested that different patterns of LTR existed in the same porcine germ line and partial PERV-C sequence may recombine with PERV-A sequence in LTR. (c) 2008 Elsevier Ltd. All rights reserved.

“Inclonals”

PubMed Central

Hakim, Rahely

2009-01-01

Full-length antibodies and antibodies that ferry a cargo to target cells are desired biopharmaceuticals. We describe the production of full-length IgGs and IgG-toxin fusion proteins in E. coli. In the presented examples of anti CD30 and anti EGF-receptor antibodies, the antibody heavy and light chains or toxin fusions thereof were expressed in separate bacterial cultures, where they accumulated as insoluble inclusion bodies. Following refolding and purification, high yields (up to 50 mg/L of shake flask culture) of highly purified (>90%) full-length antibodies and antibody-toxin fusions were obtained. The bacterially produced antibodies, named “Inclonals,” equaled the performance of the same IgGs that were produced using conventional mammalian cell culture in binding properties as well as in cell killing potency. The rapid and cost effective IgG production process and the high quality of the resultant product may make the bacterial production of full-length IgG and IgG-drug fusion proteins an attractive option for antibody production and a significant contribution to recombinant antibody technology. PMID:20065645

The giant protein titin regulates the length of the striated muscle thick filament.

PubMed

Tonino, Paola; Kiss, Balazs; Strom, Josh; Methawasin, Mei; Smith, John E; Kolb, Justin; Labeit, Siegfried; Granzier, Henk

2017-10-19

The contractile machinery of heart and skeletal muscles has as an essential component the thick filament, comprised of the molecular motor myosin. The thick filament is of a precisely controlled length, defining thereby the force level that muscles generate and how this force varies with muscle length. It has been speculated that the mechanism by which thick filament length is controlled involves the giant protein titin, but no conclusive support for this hypothesis exists. Here we show that in a mouse model in which we deleted two of titin's C-zone super-repeats, thick filament length is reduced in cardiac and skeletal muscles. In addition, functional studies reveal reduced force generation and a dilated cardiomyopathy (DCM) phenotype. Thus, regulation of thick filament length depends on titin and is critical for maintaining muscle health.

Characterization and chromosomal mapping of the human TFG gene involved in thyroid carcinoma

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mencinger, M.; Panagopoulos, I.; Andreasson, P.

1997-05-01

Homology searches in the Expressed Sequence Tag Database were performed using SPYGQ-rich regions as query sequences to find genes encoding protein regions similar to the N-terminal parts of the sarcoma-associated EWS and FUS proteins. Clone 22911 (T74973), encoding a SPYGQ-rich region in its 5{prime} end, and several other clones that overlapped 22911 were selected. The combined data made it possible to assemble a full-length cDNA sequence. This cDNA sequence is 1677 bp, containing an initiation codon ATG, an open reading frame of 400 amino acids, a poly(A) signal, and a poly(A) tail. We found 100% identity between the 5{prime} partmore » of the consensus sequence and the 598-bp-long sequence named TFG. The TFG sequence is fused to the 3{prime} end of NTRK1, generating the TRK-T3 fusion transcript found in papillary thyroid carcinoma. The cDNA therefore represents the full-length transcript of the TFG gene. TFG was localized to 3q11-q12 by fluorescence in situ hybridization. The 3{prime} and the 5{prime} ends of the TFG cDNA probe hybridized to a 2.2-kb band on Northern blot filters in all tissues examined. 28 refs., 5 figs., 1 tab.« less

Oxidative stress reduces levels of dysbindin-1A via its PEST domain.

PubMed

Yap, Mei-Yi Alicia; Lo, Yew-Long; Talbot, Konrad; Ong, Wei-Yi

2014-12-01

Oxidative stress resulting from the generation of reactive oxygen species has been proposed as an etiological factor in schizophrenia. The present study tests the hypothesis that oxidative stress can affect levels of dysbindin-1A, encoded by Dtnbp1, a genetic risk factor for schizophrenia, via its PEST domain. In vitro studies on SH-SY5Y cells indicate that oxidative stress triggers proteasomal degradation of dysbindin-1A, and that this requires interactions with its PEST domain, which may be a TRIM32 target. We specifically found (a) that oxidative stress induced in SH-SY5Y cells by 500 µM hydrogen peroxide reduced levels of full-length dysbindin-1, but did not reduce levels of that protein lacking its PEST domain and (b) that levels of full-length dysbindin-1, but not dysbindin-1 lacking its PEST domain, were higher in cells treated with the proteasome inhibitor MG132. Oxidative stress thus emerges as the first known cellular factor regulating dysbindin-1 isoforms with PEST domains. These findings are consistent with the previously noted fact that phosphorylation of PEST domains often marks proteins for proteasomal degradation, and raises the possibility that treatments reducing oxidative stress in the brain, especially during development, may lower schizophrenia risk. Copyright © 2014 Elsevier Ltd. All rights reserved.

Targeting Nonsense Mutations in Diseases with Translational Read-Through-Inducing Drugs (TRIDs).

PubMed

Nagel-Wolfrum, Kerstin; Möller, Fabian; Penner, Inessa; Baasov, Timor; Wolfrum, Uwe

2016-04-01

In recent years, remarkable advances in the ability to diagnose genetic disorders have been made. The identification of disease-causing genes allows the development of gene-specific therapies with the ultimate goal to develop personalized medicines for each patient according to their own specific genetic defect. In-depth genotyping of many different genes has revealed that ~12% of inherited genetic disorders are caused by in-frame nonsense mutations. Nonsense (non-coding) mutations are caused by point mutations, which generate premature termination codons (PTCs) that cause premature translational termination of the mRNA, and subsequently inhibit normal full-length protein expression. Recently, a gene-based therapeutic approach for genetic diseases caused by nonsense mutations has emerged, namely the so-called translational read-through (TR) therapy. Read-through therapy is based on the discovery that small molecules, known as TR-inducing drugs (TRIDs), allow the translation machinery to suppress a nonsense codon, elongate the nascent peptide chain, and consequently result in the synthesis of full-length protein. Several TRIDs are currently under investigation and research has been performed on several genetic disorders caused by nonsense mutations over the years. These findings have raised hope for the usage of TR therapy as a gene-based pharmacogenetic therapy for nonsense mutations in various genes responsible for a variety of genetic diseases.

TALEN-engineered AR gene rearrangements reveal endocrine uncoupling of androgen receptor in prostate cancer

PubMed Central

Nyquist, Michael D.; Li, Yingming; Hwang, Tae Hyun; Manlove, Luke S.; Vessella, Robert L.; Silverstein, Kevin A. T.; Voytas, Daniel F.; Dehm, Scott M.

2013-01-01

Androgen receptor (AR) target genes direct development and survival of the prostate epithelial lineage, including prostate cancer (PCa). Thus, endocrine therapies that inhibit the AR ligand-binding domain (LBD) are effective in treating PCa. AR transcriptional reactivation is central to resistance, as evidenced by the efficacy of AR retargeting in castration-resistant PCa (CRPC) with next-generation endocrine therapies abiraterone and enzalutamide. However, resistance to abiraterone and enzalutamide limits this efficacy in most men, and PCa remains the second-leading cause of male cancer deaths. Here we show that AR gene rearrangements in CRPC tissues underlie a completely androgen-independent, yet AR-dependent, resistance mechanism. We discovered intragenic AR gene rearrangements in CRPC tissues, which we modeled using transcription activator-like effector nuclease (TALEN)-mediated genome engineering. This modeling revealed that these AR gene rearrangements blocked full-length AR synthesis, but promoted expression of truncated AR variant proteins lacking the AR ligand-binding domain. Furthermore, these AR variant proteins maintained the constitutive activity of the AR transcriptional program and a CRPC growth phenotype independent of full-length AR or androgens. These findings demonstrate that AR gene rearrangements are a unique resistance mechanism by which AR transcriptional activity can be uncoupled from endocrine regulation in CRPC. PMID:24101480

The P0 protein encoded by cotton leafroll dwarf virus (CLRDV) inhibits local but not systemic RNA silencing.

PubMed

Delfosse, Verónica C; Agrofoglio, Yamila C; Casse, María F; Kresic, Iván Bonacic; Hopp, H Esteban; Ziegler-Graff, Véronique; Distéfano, Ana J

2014-02-13

Plants employ RNA silencing as a natural defense mechanism against viruses. As a counter-defense, viruses encode silencing suppressor proteins (SSPs) that suppress RNA silencing. Most, but not all, the P0 proteins encoded by poleroviruses have been identified as SSP. In this study, we demonstrated that cotton leafroll dwarf virus (CLRDV, genus Polerovirus) P0 protein suppressed local silencing that was induced by sense or inverted repeat transgenes in Agrobacterium co-infiltration assay in Nicotiana benthamiana plants. A CLRDV full-length infectious cDNA clone that is able to infect N. benthamiana through Agrobacterium-mediated inoculation also inhibited local silencing in co-infiltration assays, suggesting that the P0 protein exhibits similar RNA silencing suppression activity when expressed from the full-length viral genome. On the other hand, the P0 protein did not efficiently inhibit the spread of systemic silencing signals. Moreover, Northern blotting indicated that the P0 protein inhibits the generation of secondary but not primary small interfering RNAs. The study of CLRDV P0 suppression activity may contribute to understanding the molecular mechanisms involved in the induction of cotton blue disease by CLRDV infection. Copyright © 2013 Elsevier B.V. All rights reserved.

MINIGENOMES, TRANSCRIPTION AND REPLICATION COMPETENT VIRUS-LIKE PARTICLES AND BEYOND: REVERSE GENETICS SYSTEMS FOR FILOVIRUSES AND OTHER NEGATIVE STRANDED HEMORRHAGIC FEVER VIRUSES

PubMed Central

Hoenen, Thomas; Groseth, Allison; de Kok-Mercado, Fabian; Kuhn, Jens H.; Wahl-Jensen, Victoria

2012-01-01

Reverse-genetics systems are powerful tools enabling researchers to study the replication cycle of RNA viruses, including filoviruses and other hemorrhagic fever viruses, as well as to discover new antivirals. They include full-length clone systems as well as a number of life cycle modeling systems. Full-length clone systems allow for the generation of infectious, recombinant viruses, and thus are an important tool for studying the virus replication cycle in its entirety. In contrast, life cycle modeling systems such as minigenome and transcription and replication competent virus-like particle systems can be used to simulate and dissect parts of the virus life cycle outside of containment facilities. Minigenome systems are used to model viral genome replication and transcription, whereas transcription and replication competent virus-like particle systems also model morphogenesis and budding as well as infection of target cells. As such, these modeling systems have tremendous potential to further the discovery and screening of new antivirals targeting hemorrhagic fever viruses. This review provides an overview of currently established reverse genetics systems for hemorrhagic fever-causing negative-sense RNA viruses, with a particular emphasis on filoviruses, and the potential application of these systems for antiviral research. PMID:21699921

Recombination in Avian Gamma-Coronavirus Infectious Bronchitis Virus

PubMed Central

Thor, Sharmi W.; Hilt, Deborah A.; Kissinger, Jessica C.; Paterson, Andrew H.; Jackwood, Mark W.

2011-01-01

Recombination in the family Coronaviridae has been well documented and is thought to be a contributing factor in the emergence and evolution of different coronaviral genotypes as well as different species of coronavirus. However, there are limited data available on the frequency and extent of recombination in coronaviruses in nature and particularly for the avian gamma-coronaviruses where only recently the emergence of a turkey coronavirus has been attributed solely to recombination. In this study, the full-length genomes of eight avian gamma-coronavirus infectious bronchitis virus (IBV) isolates were sequenced and along with other full-length IBV genomes available from GenBank were analyzed for recombination. Evidence of recombination was found in every sequence analyzed and was distributed throughout the entire genome. Areas that have the highest occurrence of recombination are located in regions of the genome that code for nonstructural proteins 2, 3 and 16, and the structural spike glycoprotein. The extent of the recombination observed, suggests that this may be one of the principal mechanisms for generating genetic and antigenic diversity within IBV. These data indicate that reticulate evolutionary change due to recombination in IBV, likely plays a major role in the origin and adaptation of the virus leading to new genetic types and strains of the virus. PMID:21994806

Allosteric alterations in the androgen receptor and activity in prostate cancer.

PubMed

Uo, Takuma; Plymate, Stephen R; Sprenger, Cynthia C

2017-09-01

Organisms have evolved to generate biological complexity in their proteome and transcriptome from a limited number of genes. This concept holds true for the androgen receptor, which displays a diversity of inclusion/exclusion events in its structural motifs as a mechanism of resistance to the most forefront anti-androgen therapies. More than 20 androgen receptor variants that lack various portions of ligand-binding domain have been identified in human prostate cancer (PCa) samples. Most of the variants are inactive on their own, with a few exceptions displaying constitutive activity. The full-length receptor and one or more variants can be co-expressed in the same cell under many circumstances, which raises the question of how these variants physically and functionally interact with the full-length receptor or one another in the course of PCa progression. To address this issue, in this review, we will characterize and discuss androgen receptor variants, including the novel variants discovered in the last couple of years (i) individually, (ii) with respect to their physical and functional interaction with one another and (iii) in clinical relevance. Here, we also introduce the very recent understanding of AR-Vs obtained through successful development of some AR-V-specific antibodies as well as identification of novel AR-Vs by data mining approaches. © 2017 Society for Endocrinology.

Integrating De Novo Transcriptome Assembly and Cloning to Obtain Chicken Ovocleidin-17 Full-Length cDNA

PubMed Central

Ning, ZhongHua; Hincke, Maxwell T.; Yang, Ning; Hou, ZhuoCheng

2014-01-01

Efficiently obtaining full-length cDNA for a target gene is the key step for functional studies and probing genetic variations. However, almost all sequenced domestic animal genomes are not ‘finished’. Many functionally important genes are located in these gapped regions. It can be difficult to obtain full-length cDNA for which only partial amino acid/EST sequences exist. In this study we report a general pipeline to obtain full-length cDNA, and illustrate this approach for one important gene (Ovocleidin-17, OC-17) that is associated with chicken eggshell biomineralization. Chicken OC-17 is one of the best candidates to control and regulate the deposition of calcium carbonate in the calcified eggshell layer. OC-17 protein has been purified, sequenced, and has had its three-dimensional structure solved. However, researchers still cannot conduct OC-17 mRNA related studies because the mRNA sequence is unknown and the gene is absent from the current chicken genome. We used RNA-Seq to obtain the entire transcriptome of the adult hen uterus, and then conducted de novo transcriptome assembling with bioinformatics analysis to obtain candidate OC-17 transcripts. Based on this sequence, we used RACE and PCR cloning methods to successfully obtain the full-length OC-17 cDNA. Temporal and spatial OC-17 mRNA expression analyses were also performed to demonstrate that OC-17 is predominantly expressed in the adult hen uterus during the laying cycle and barely at immature developmental stages. Differential uterine expression of OC-17 was observed in hens laying eggs with weak versus strong eggshell, confirming its important role in the regulation of eggshell mineralization and providing a new tool for genetic selection for eggshell quality parameters. This study is the first one to report the full-length OC-17 cDNA sequence, and builds a foundation for OC-17 mRNA related studies. We provide a general method for biologists experiencing difficulty in obtaining candidate gene full-length cDNA sequences. PMID:24676480

Integrating de novo transcriptome assembly and cloning to obtain chicken Ovocleidin-17 full-length cDNA.

PubMed

Zhang, Quan; Liu, Long; Zhu, Feng; Ning, ZhongHua; Hincke, Maxwell T; Yang, Ning; Hou, ZhuoCheng

2014-01-01

Efficiently obtaining full-length cDNA for a target gene is the key step for functional studies and probing genetic variations. However, almost all sequenced domestic animal genomes are not 'finished'. Many functionally important genes are located in these gapped regions. It can be difficult to obtain full-length cDNA for which only partial amino acid/EST sequences exist. In this study we report a general pipeline to obtain full-length cDNA, and illustrate this approach for one important gene (Ovocleidin-17, OC-17) that is associated with chicken eggshell biomineralization. Chicken OC-17 is one of the best candidates to control and regulate the deposition of calcium carbonate in the calcified eggshell layer. OC-17 protein has been purified, sequenced, and has had its three-dimensional structure solved. However, researchers still cannot conduct OC-17 mRNA related studies because the mRNA sequence is unknown and the gene is absent from the current chicken genome. We used RNA-Seq to obtain the entire transcriptome of the adult hen uterus, and then conducted de novo transcriptome assembling with bioinformatics analysis to obtain candidate OC-17 transcripts. Based on this sequence, we used RACE and PCR cloning methods to successfully obtain the full-length OC-17 cDNA. Temporal and spatial OC-17 mRNA expression analyses were also performed to demonstrate that OC-17 is predominantly expressed in the adult hen uterus during the laying cycle and barely at immature developmental stages. Differential uterine expression of OC-17 was observed in hens laying eggs with weak versus strong eggshell, confirming its important role in the regulation of eggshell mineralization and providing a new tool for genetic selection for eggshell quality parameters. This study is the first one to report the full-length OC-17 cDNA sequence, and builds a foundation for OC-17 mRNA related studies. We provide a general method for biologists experiencing difficulty in obtaining candidate gene full-length cDNA sequences.

Effects of complex life cycles on genetic diversity: cyclical parthenogenesis.

PubMed

Rouger, R; Reichel, K; Malrieu, F; Masson, J P; Stoeckel, S

2016-11-01

Neutral patterns of population genetic diversity in species with complex life cycles are difficult to anticipate. Cyclical parthenogenesis (CP), in which organisms undergo several rounds of clonal reproduction followed by a sexual event, is one such life cycle. Many species, including crop pests (aphids), human parasites (trematodes) or models used in evolutionary science (Daphnia), are cyclical parthenogens. It is therefore crucial to understand the impact of such a life cycle on neutral genetic diversity. In this paper, we describe distributions of genetic diversity under conditions of CP with various clonal phase lengths. Using a Markov chain model of CP for a single locus and individual-based simulations for two loci, our analysis first demonstrates that strong departures from full sexuality are observed after only a few generations of clonality. The convergence towards predictions made under conditions of full clonality during the clonal phase depends on the balance between mutations and genetic drift. Second, the sexual event of CP usually resets the genetic diversity at a single locus towards predictions made under full sexuality. However, this single recombination event is insufficient to reshuffle gametic phases towards full-sexuality predictions. Finally, for similar levels of clonality, CP and acyclic partial clonality (wherein a fixed proportion of individuals are clonally produced within each generation) differentially affect the distribution of genetic diversity. Overall, this work provides solid predictions of neutral genetic diversity that may serve as a null model in detecting the action of common evolutionary or demographic processes in cyclical parthenogens (for example, selection or bottlenecks).

Realistic modeling of deep brain stimulation implants for electromagnetic MRI safety studies.

PubMed

Guerin, Bastien; Serano, Peter; Iacono, Maria Ida; Herrington, Todd M; Widge, Alik S; Dougherty, Darin D; Bonmassar, Giorgio; Angelone, Leonardo M; Wald, Lawrence L

2018-05-04

We propose a framework for electromagnetic (EM) simulation of deep brain stimulation (DBS) patients in radiofrequency (RF) coils. We generated a model of a DBS patient using post-operative head and neck computed tomography (CT) images stitched together into a 'virtual CT' image covering the entire length of the implant. The body was modeled as homogeneous. The implant path extracted from the CT data contained self-intersections, which we corrected automatically using an optimization procedure. Using the CT-derived DBS path, we built a model of the implant including electrodes, helicoidal internal conductor wires, loops, extension cables, and the implanted pulse generator. We also built four simplified models with straight wires, no extension cables and no loops to assess the impact of these simplifications on safety predictions. We simulated EM fields induced by the RF birdcage body coil in the body model, including at the DBS lead tip at both 1.5 Tesla (64 MHz) and 3 Tesla (123 MHz). We also assessed the robustness of our simulation results by systematically varying the EM properties of the body model and the position and length of the DBS implant (sensitivity analysis). The topology correction algorithm corrected all self-intersection and curvature violations of the initial path while introducing minimal deformations (open-source code available at http://ptx.martinos.org/index.php/Main_Page). The unaveraged lead-tip peak SAR predicted by the five DBS models (0.1 mm resolution grid) ranged from 12.8 kW kg -1 (full model, helicoidal conductors) to 43.6 kW kg -1 (no loops, straight conductors) at 1.5 T (3.4-fold variation) and 18.6 kW kg -1 (full model, straight conductors) to 73.8 kW kg -1 (no loops, straight conductors) at 3 T (4.0-fold variation). At 1.5 T and 3 T, the variability of lead-tip peak SAR with respect to the conductivity ranged between 18% and 30%. Variability with respect to the position and length of the DBS implant ranged between 9.5% and 27.6%.

Realistic modeling of deep brain stimulation implants for electromagnetic MRI safety studies

NASA Astrophysics Data System (ADS)

Guerin, Bastien; Serano, Peter; Iacono, Maria Ida; Herrington, Todd M.; Widge, Alik S.; Dougherty, Darin D.; Bonmassar, Giorgio; Angelone, Leonardo M.; Wald, Lawrence L.

2018-05-01

We propose a framework for electromagnetic (EM) simulation of deep brain stimulation (DBS) patients in radiofrequency (RF) coils. We generated a model of a DBS patient using post-operative head and neck computed tomography (CT) images stitched together into a ‘virtual CT’ image covering the entire length of the implant. The body was modeled as homogeneous. The implant path extracted from the CT data contained self-intersections, which we corrected automatically using an optimization procedure. Using the CT-derived DBS path, we built a model of the implant including electrodes, helicoidal internal conductor wires, loops, extension cables, and the implanted pulse generator. We also built four simplified models with straight wires, no extension cables and no loops to assess the impact of these simplifications on safety predictions. We simulated EM fields induced by the RF birdcage body coil in the body model, including at the DBS lead tip at both 1.5 Tesla (64 MHz) and 3 Tesla (123 MHz). We also assessed the robustness of our simulation results by systematically varying the EM properties of the body model and the position and length of the DBS implant (sensitivity analysis). The topology correction algorithm corrected all self-intersection and curvature violations of the initial path while introducing minimal deformations (open-source code available at http://ptx.martinos.org/index.php/Main_Page). The unaveraged lead-tip peak SAR predicted by the five DBS models (0.1 mm resolution grid) ranged from 12.8 kW kg‑1 (full model, helicoidal conductors) to 43.6 kW kg‑1 (no loops, straight conductors) at 1.5 T (3.4-fold variation) and 18.6 kW kg‑1 (full model, straight conductors) to 73.8 kW kg‑1 (no loops, straight conductors) at 3 T (4.0-fold variation). At 1.5 T and 3 T, the variability of lead-tip peak SAR with respect to the conductivity ranged between 18% and 30%. Variability with respect to the position and length of the DBS implant ranged between 9.5% and 27.6%.

Genetic deletion of muscle RANK or selective inhibition of RANKL is not as effective as full-length OPG-fc in mitigating muscular dystrophy.

PubMed

Dufresne, Sébastien S; Boulanger-Piette, Antoine; Bossé, Sabrina; Argaw, Anteneh; Hamoudi, Dounia; Marcadet, Laetitia; Gamu, Daniel; Fajardo, Val A; Yagita, Hideo; Penninger, Josef M; Russell Tupling, A; Frenette, Jérôme

2018-04-24

Although there is a strong association between osteoporosis and skeletal muscle atrophy/dysfunction, the functional relevance of a particular biological pathway that regulates synchronously bone and skeletal muscle physiopathology is still elusive. Receptor-activator of nuclear factor κB (RANK), its ligand RANKL and the soluble decoy receptor osteoprotegerin (OPG) are the key regulators of osteoclast differentiation and bone remodelling. We thus hypothesized that RANK/RANKL/OPG, which is a key pathway for bone regulation, is involved in Duchenne muscular dystrophy (DMD) physiopathology. Our results show that muscle-specific RANK deletion (mdx-RANK mko ) in dystrophin deficient mdx mice improves significantly specific force [54% gain in force] of EDL muscles with no protective effect against eccentric contraction-induced muscle dysfunction. In contrast, full-length OPG-Fc injections restore the force of dystrophic EDL muscles [162% gain in force], protect against eccentric contraction-induced muscle dysfunction ex vivo and significantly improve functional performance on downhill treadmill and post-exercise physical activity. Since OPG serves a soluble receptor for RANKL and as a decoy receptor for TRAIL, mdx mice were injected with anti-RANKL and anti-TRAIL antibodies to decipher the dual function of OPG. Injections of anti-RANKL and/or anti-TRAIL increase significantly the force of dystrophic EDL muscle [45% and 17% gains in force, respectively]. In agreement, truncated OPG-Fc that contains only RANKL domains produces similar gains, in terms of force production, than anti-RANKL treatments. To corroborate that full-length OPG-Fc also acts independently of RANK/RANKL pathway, dystrophin/RANK double-deficient mice were treated with full-length OPG-Fc for 10 days. Dystrophic EDL muscles exhibited a significant gain in force relative to untreated dystrophin/RANK double-deficient mice, indicating that the effect of full-length OPG-Fc is in part independent of the RANKL/RANK interaction. The sarco/endoplasmic reticulum Ca 2+ ATPase (SERCA) activity is significantly depressed in dysfunctional and dystrophic muscles and full-length OPG-Fc treatment increased SERCA activity and SERCA-2a expression. These findings demonstrate the superiority of full-length OPG-Fc treatment relative to truncated OPG-Fc, anti-RANKL, anti-TRAIL or muscle RANK deletion in improving dystrophic muscle function, integrity and protection against eccentric contractions. In conclusion, full-length OPG-Fc represents an efficient alternative in the development of new treatments for muscular dystrophy in which a single therapeutic approach may be foreseeable to maintain both bone and skeletal muscle functions.

Effect of six types of footwear on peak plantar pressures in patients with diabetes and transmetatarsal amputation.

PubMed

Mueller, MJ; Strube, MJ; Allen, BT

1997-04-01

INTRODUCTION:: Patients with diabetes (DM) and transmetatarsal amputation (TMA) are at high risk for skin breakdown from excessive peak plantar pressures (PPP). The primary purpose of this study was to determine how footwear (full length shoe or short shoe), a total contact insert, a rigid-rocker bottom (RRB) sole, and an ankle-foot-orthosis (AFO) affect PPP on the distal residuum and contralateral extremity of patients with DM and TMA. A secondary purpose was to monitor various functional measures during use of the footwear. METHODS:: Thirty patients with DM and TMA participated (mean age 62+/-4 years). The mean duration of DM was 19.9+/-10.1 years, and the mean time since TMA was 27.4+/-28.1 months. The following footwear was provided after a check-out from an orthotist and physical therapist (PT); 1) Full length shoe (ie shoe length prior to surgery), with a toe filler, 2) full length shoe, total contact insert, and an AFO, 2) full length shoe, total contact insert, and an AFO, 3) full length shoe, total contact insert, and a RRB sole, 4) full length shoe, total contact insert, RRB sole, and an AFO, 5) short shoe (ie length of residuum), total contact insert, and RRB, 6) short shoe, total contact insert, AFO, and RRB sole. In-shoe PPP during walking at the distal residuum and forefoot of the contralateral extremity were measured using the F-Scan System with established reliability under similar conditions (Generilizability coefficient =.75). Each measurement occurred after a one month adjustment period. Data were analyzed using a univariate repeated measuresANOVA. Individual contrasts were used for post-hoc analysis on those variables showing a significant overall F value (p<.05). RESULTS:: Compared to a regular shoe with a toe-filler, all conditions except the short shoe (#5), resulted in lower PPP on the distal residuum (p<.05). Condition 3, the full length shoe, total contact insert, and RRB resulted in lower pressures on the distal residuum and forefoot of the contralateral extremity compared to a regular shoe and toe-filler, and had few functional complaints as identified by the patient, orthotist or PT (3/27). Footwear using an AFO (Conditions 2,4,6) showed reduced PPP on the residuum, but most patients (16/29) had functional complaints. The short shoe (condition 5) had the fewest[Table: see text] functional complaints (2/26), but did not significantly reduce PPP and had the highest cosmetic refusal rate (5/26). DISCUSSION AND CONCLUSIONS:: Although there are individual patient characteristics which warrant other prescriptions, based on the results of this study, we recommend the full length shoe, total contact insert, and RRB sole for most patients with DM and TMA to reduce PPP. A reduction in PPP should help to lower the high risk of skin breakdown in this patient population.

X-ray laser system, x-ray laser and method

DOEpatents

London, Richard A.; Rosen, Mordecai D.; Strauss, Moshe

1992-01-01

Disclosed is an x-ray laser system comprising a laser containing generating means for emitting short wave length radiation, and means external to said laser for energizing said generating means, wherein when the laser is in an operative mode emitting radiation, the radiation has a transverse coherence length to width ratio of from about 0.05 to 1. Also disclosed is a method of adjusting the parameters of the laser to achieve the desired coherence length to laser width ratio.

Probabilistic Description of Fatigue Crack Growth Under Constant-and Variable-Amplitude Loading

DTIC Science & Technology

1989-03-01

plane, see figure 14. The length of the defected crack component and its angle, b and q, respectively, in Figure 15 were found to depend on the crack...length at which the defection occurs; as the crack length increases, b increases while q decreases. Due to the orientation of the deflected component...Breakpoint Voltage to Fun. Generator Output Setpoint Voltage Take Function Generator Gate High Start Test LNext page 153 Q! ~From last ag lastr DMAe 70

piggyBac transposons expressing full-length human dystrophin enable genetic correction of dystrophic mesoangioblasts

PubMed Central

Loperfido, Mariana; Jarmin, Susan; Dastidar, Sumitava; Di Matteo, Mario; Perini, Ilaria; Moore, Marc; Nair, Nisha; Samara-Kuko, Ermira; Athanasopoulos, Takis; Tedesco, Francesco Saverio; Dickson, George; Sampaolesi, Maurilio; VandenDriessche, Thierry; Chuah, Marinee K.

2016-01-01

Duchenne muscular dystrophy (DMD) is a genetic neuromuscular disorder caused by the absence of dystrophin. We developed a novel gene therapy approach based on the use of the piggyBac (PB) transposon system to deliver the coding DNA sequence (CDS) of either full-length human dystrophin (DYS: 11.1 kb) or truncated microdystrophins (MD1: 3.6 kb; MD2: 4 kb). PB transposons encoding microdystrophins were transfected in C2C12 myoblasts, yielding 65±2% MD1 and 66±2% MD2 expression in differentiated multinucleated myotubes. A hyperactive PB (hyPB) transposase was then deployed to enable transposition of the large-size PB transposon (17 kb) encoding the full-length DYS and green fluorescence protein (GFP). Stable GFP expression attaining 78±3% could be achieved in the C2C12 myoblasts that had undergone transposition. Western blot analysis demonstrated expression of the full-length human DYS protein in myotubes. Subsequently, dystrophic mesoangioblasts from a Golden Retriever muscular dystrophy dog were transfected with the large-size PB transposon resulting in 50±5% GFP-expressing cells after stable transposition. This was consistent with correction of the differentiated dystrophic mesoangioblasts following expression of full-length human DYS. These results pave the way toward a novel non-viral gene therapy approach for DMD using PB transposons underscoring their potential to deliver large therapeutic genes. PMID:26682797

Studies of nontarget-mediated distribution of human full-length IgG1 antibody and its FAb fragment in cardiovascular and metabolic-related tissues.

PubMed

Davidsson, Pia; Söderling, Ann-Sofi; Svensson, Lena; Ahnmark, Andrea; Flodin, Christine; Wanag, Ewa; Screpanti-Sundqvist, Valentina; Gennemark, Peter

2015-05-01

Tissue distribution and pharmacokinetics (PK) of full-length nontargeted antibody and its antigen-binding fragment (FAb) were evaluated for a range of tissues primarily of interest for cardiovascular and metabolic diseases. Mice were intravenously injected with a dose of 10 mg/kg of either human IgG1or its FAb fragment; perfused tissues were collected at a range of time points over 3 weeks for the human IgG1 antibody and 1 week for the human FAb antibody. Tissues were homogenized and antibody concentrations were measured by specific immunoassays on the Gyros system. Exposure in terms of maximum concentration (Cmax ) and area under the curve was assessed for all nine tissues. Tissue exposure of full-length antibody relative to plasma exposure was found to be between 1% and 10%, except for brain (0.2%). Relative concentrations of FAb antibody were the same, except for kidney tissue, where the antibody concentration was found to be ten times higher than in plasma. However, the absolute tissue uptake of full-length IgG was significantly higher than the absolute tissue uptake of the FAb antibody. This study provides a reference PK state for full-length whole and FAb antibodies in tissues related to cardiovascular and metabolic diseases that do not include antigen or antibody binding. © 2015 Wiley Periodicals, Inc. and the American Pharmacists Association.

Regulation of presynaptic Ca2+, synaptic plasticity and contextual fear conditioning by a N-terminal β-amyloid fragment.

PubMed

Lawrence, James L M; Tong, Mei; Alfulaij, Naghum; Sherrin, Tessi; Contarino, Mark; White, Michael M; Bellinger, Frederick P; Todorovic, Cedomir; Nichols, Robert A

2014-10-22

Soluble β-amyloid has been shown to regulate presynaptic Ca(2+) and synaptic plasticity. In particular, picomolar β-amyloid was found to have an agonist-like action on presynaptic nicotinic receptors and to augment long-term potentiation (LTP) in a manner dependent upon nicotinic receptors. Here, we report that a functional N-terminal domain exists within β-amyloid for its agonist-like activity. This sequence corresponds to a N-terminal fragment generated by the combined action of α- and β-secretases, and resident carboxypeptidase. The N-terminal β-amyloid fragment is present in the brains and CSF of healthy adults as well as in Alzheimer's patients. Unlike full-length β-amyloid, the N-terminal β-amyloid fragment is monomeric and nontoxic. In Ca(2+) imaging studies using a model reconstituted rodent neuroblastoma cell line and isolated mouse nerve terminals, the N-terminal β-amyloid fragment proved to be highly potent and more effective than full-length β-amyloid in its agonist-like action on nicotinic receptors. In addition, the N-terminal β-amyloid fragment augmented theta burst-induced post-tetanic potentiation and LTP in mouse hippocampal slices. The N-terminal fragment also rescued LTP inhibited by elevated levels of full-length β-amyloid. Contextual fear conditioning was also strongly augmented following bilateral injection of N-terminal β-amyloid fragment into the dorsal hippocampi of intact mice. The fragment-induced augmentation of fear conditioning was attenuated by coadministration of nicotinic antagonist. The activity of the N-terminal β-amyloid fragment appears to reside largely in a sequence surrounding a putative metal binding site, YEVHHQ. These findings suggest that the N-terminal β-amyloid fragment may serve as a potent and effective endogenous neuromodulator. Copyright © 2014 the authors 0270-6474/14/3414210-09$15.00/0.

Sensitivity of a computer adaptive assessment for measuring functional mobility changes in children enrolled in a community fitness programme.

PubMed

Haley, Stephen M; Fragala-Pinkham, Maria; Ni, Pengsheng

2006-07-01

To examine the relative sensitivity to detect functional mobility changes with a full-length parent questionnaire compared with a computerized adaptive testing version of the questionnaire after a 16-week group fitness programme. Prospective, pre- and posttest study with a 16-week group fitness intervention. Three community-based fitness centres. Convenience sample of children (n = 28) with physical or developmental disabilities. A 16-week group exercise programme held twice a week in a community setting. A full-length (161 items) paper version of a mobility parent questionnaire based on the Pediatric Evaluation of Disability Inventory, but expanded to include expected skills of children up to 15 years old was compared with a 15-item computer adaptive testing version. Both measures were administered at pre- and posttest intervals. Both the full-length Pediatric Evaluation of Disability Inventory and the 15-item computer adaptive testing version detected significant changes between pre- and posttest scores, had large effect sizes, and standardized response means, with a modest decrease in the computer adaptive test as compared with the 161-item paper version. Correlations between the computer adaptive and paper formats across pre- and posttest scores ranged from r = 0.76 to 0.86. Both functional mobility test versions were able to detect positive functional changes at the end of the intervention period. Greater variability in score estimates was generated by the computerized adaptive testing version, which led to a relative reduction in sensitivity as defined by the standardized response mean. Extreme scores were generally more difficult for the computer adaptive format to estimate with as much accuracy as scores in the mid-range of the scale. However, the reduction in accuracy and sensitivity, which did not influence the group effect results in this study, is counterbalanced by the large reduction in testing burden.

A Novel mouse model of enhanced proteostasis: Full-length human heat shock factor 1 transgenic mice

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pierce, Anson, E-mail: piercea2@uthscsa.edu; Barshop Institute for Longevity and Aging Studies, The University of Texas Health Science Center at San Antonio, San Antonio, Texas, 78229; The Department of Veteran's Affairs, South Texas Veterans Health Care System, San Antonio, Texas, 78284

2010-11-05

Research highlights: {yields} Development of mouse overexpressing native human HSF1 in all tissues including CNS. {yields} HSF1 overexpression enhances heat shock response at whole-animal and cellular level. {yields} HSF1 overexpression protects from polyglutamine toxicity and favors aggresomes. {yields} HSF1 overexpression enhances proteostasis at the whole-animal and cellular level. -- Abstract: The heat shock response (HSR) is controlled by the master transcriptional regulator heat shock factor 1 (HSF1). HSF1 maintains proteostasis and resistance to stress through production of heat shock proteins (HSPs). No transgenic model exists that overexpresses HSF1 in tissues of the central nervous system (CNS). We generated a transgenicmore » mouse overexpressing full-length non-mutant HSF1 and observed a 2-4-fold increase in HSF1 mRNA and protein expression in all tissues studied of HSF1 transgenic (HSF1{sup +/0}) mice compared to wild type (WT) littermates, including several regions of the CNS. Basal expression of HSP70 and 90 showed only mild tissue-specific changes; however, in response to forced exercise, the skeletal muscle HSR was more elevated in HSF1{sup +/0} mice compared to WT littermates and in fibroblasts following heat shock, as indicated by levels of inducible HSP70 mRNA and protein. HSF1{sup +/0} cells elicited a significantly more robust HSR in response to expression of the 82 repeat polyglutamine-YFP fusion construct (Q82YFP) and maintained proteasome-dependent processing of Q82YFP compared to WT fibroblasts. Overexpression of HSF1 was associated with fewer, but larger Q82YFP aggregates resembling aggresomes in HSF1{sup +/0} cells, and increased viability. Therefore, our data demonstrate that tissues and cells from mice overexpressing full-length non-mutant HSF1 exhibit enhanced proteostasis.« less

Biochip-based instruments development for space exploration: influence of the antibody immobilization process on the biochip resistance to freeze-drying, temperature shifts and cosmic radiations

NASA Astrophysics Data System (ADS)

Coussot, G.; Moreau, T.; Faye, C.; Vigier, F.; Baqué, M.; Le Postollec, A.; Incerti, S.; Dobrijevic, M.; Vandenabeele-Trambouze, O.

2017-04-01

Due to the diversity of antibody (Ab)-based biochips chemistries available and the little knowledge about biochips resistance to space constraints, immobilization of Abs on the surface of the biochips dedicated to Solar System exploration is challenging. In the present paper, we have developed ten different biochip models including covalent or affinity immobilization with full-length Abs or Ab fragments. Ab immobilizations were carried out in oriented/non-oriented manner using commercial activated surfaces with N-hydroxysuccinic ester (NHS-surfaces) or homemade surfaces using three generations of dendrimers (dendrigraft of poly L-lysine (DGL) surfaces). The performances of the Ab -based surfaces were cross-compared on the following criteria: (i) analytical performances (expressed by both the surface density of immobilized Abs and the amount of antigens initially captured by the surface) and (ii) resistance of surfaces to preparation procedure (freeze-drying, storage) or spatial constraints (irradiation and temperature shifts) encountered during a space mission. The latter results have been expressed as percentage of surface binding capacity losses (or percentage of remaining active Abs). The highest amount of captured antigen was achieved with Ab surfaces having full-length Abs and DGL-surfaces that have much higher surface densities than commercial NHS-surface. After freeze-drying process, thermal shift and storage sample exposition, we found that more than 80% of surface binding sites remained active in this case. In addition, the resistance of Ab surfaces to irradiation with particles such as electron, carbon ions or protons depends not only on the chemistries (covalent/affinity linkages) and strategies (oriented/non-oriented) used to construct the biochip, but also on the type, energy and fluence of incident particles. Our results clearly indicate that full-length Ab immobilization on NHS-surfaces and DGL-surfaces should be preferred for potential use in instruments for planetary exploration.

Micropropagation and assessment of genetic fidelity of Dendrocalamus strictus (Roxb.) nees using RAPD and ISSR markers.

PubMed

Goyal, Arvind Kumar; Pradhan, Sushen; Basistha, Bharat Chandra; Sen, Arnab

2015-08-01

Dendrocalamus strictus popularly known as 'Male bamboo' is a multipurpose bamboo which is extensively utilized in pharmaceutical, paper, agricultural and other industrial implements. In this study, in vitro regeneration of D. strictus through nodal culture has been attempted. Murashige and Skoog's medium supplemented with 4 mg/l BAP was found to be most effective in shoot regeneration with 3.68 ± 0.37 shoots per explant. The effect of Kn was found to be moderate. These hormones also had considerable effect on the shoot length. The highest shoot length after 6 weeks (3.11 ± 0.41 cm) was noted with 5 mg/l BAP followed by 3.07 ± 0.28 cm with 5 mg/l Kn, while decrease in the shoot length was noted with other treatments. The effect of IBA and NAA individually or in combination at different concentrations on rooting was evaluated. The highest number of root (1.36 ± 0.04) was regenerated on full-strength MS medium supplemented with 3 mg/l NAA, while maximum length of 1.64 ± 0.03 cm of roots was recorded with combination of 1 mg/l IBA and 3 mg/l NAA. Tissue-cultured plants thus obtained were successfully transferred to the soil. The clonal fidelity among the in vitro-regenerated plantlets was assessed by RAPD and ISSR markers. The ten RAPD decamers produced 58 amplicons, while nine ISSR primers generated a total of 66 bands. All the bands generated were monomorphic. These results confirmed the clonal fidelity of the tissue culture-raised D. strictus plantlets and corroborated the fact that nodal culture is perhaps the safest mode for multiplication of true to type plants.

Self-assembly Controls Self-cleavage of HHR from ASBVd (-): a Combined SANS and Modeling Study

NASA Astrophysics Data System (ADS)

Leclerc, Fabrice; Zaccai, Giuseppe; Vergne, Jacques; Řìhovà, Martina; Martel, Anne; Maurel, Marie-Christine

2016-07-01

In the Avocado Sunblotch Viroid (ASBVd: 249-nt) from the Avsunviroidae family, a symmetric rolling-circle replication operates through an autocatalytic mechanism mediated by hammerhead ribozymes (HHR) embedded in both polarity strands. The concatenated multimeric ASBVd (+) and ASBVd (-) RNAs thus generated are processed by cleavage to unit-length where ASBVd (-) self-cleaves with more efficiency. Absolute scale small angle neutron scattering (SANS) revealed a temperature-dependent dimer association in both ASBVd (-) and its derived 79-nt HHR (-). A joint thermodynamic analysis of SANS and catalytic data indicates the rate-determining step corresponds to the dimer/monomer transition. 2D and 3D models of monomeric and dimeric HHR (-) suggest that the inter-molecular contacts stabilizing the dimer (between HI and HII domains) compete with the intra-molecular ones stabilizing the active conformation of the full-length HHR required for an efficient self-cleavage. Similar competing intra- and inter-molecular contacts are proposed in ASBVd (-) though with a remoter region from an extension of the HI domain.

A new comprehensive method for detection of livestock-related pathogenic viruses using a target enrichment system.

PubMed

Oba, Mami; Tsuchiaka, Shinobu; Omatsu, Tsutomu; Katayama, Yukie; Otomaru, Konosuke; Hirata, Teppei; Aoki, Hiroshi; Murata, Yoshiteru; Makino, Shinji; Nagai, Makoto; Mizutani, Tetsuya

2018-01-08

We tested usefulness of a target enrichment system SureSelect, a comprehensive viral nucleic acid detection method, for rapid identification of viral pathogens in feces samples of cattle, pigs and goats. This system enriches nucleic acids of target viruses in clinical/field samples by using a library of biotinylated RNAs with sequences complementary to the target viruses. The enriched nucleic acids are amplified by PCR and subjected to next generation sequencing to identify the target viruses. In many samples, SureSelect target enrichment method increased efficiencies for detection of the viruses listed in the biotinylated RNA library. Furthermore, this method enabled us to determine nearly full-length genome sequence of porcine parainfluenza virus 1 and greatly increased Breadth, a value indicating the ratio of the mapping consensus length in the reference genome, in pig samples. Our data showed usefulness of SureSelect target enrichment system for comprehensive analysis of genomic information of various viruses in field samples. Copyright © 2017 Elsevier Inc. All rights reserved.

Discovery of a novel iflavirus sequence in the eastern paralysis tick Ixodes holocyclus.

PubMed

O'Brien, Caitlin A; Hall-Mendelin, Sonja; Hobson-Peters, Jody; Deliyannis, Georgia; Allen, Andy; Lew-Tabor, Ala; Rodriguez-Valle, Manuel; Barker, Dayana; Barker, Stephen C; Hall, Roy A

2018-05-11

Ixodes holocyclus, the eastern paralysis tick, is a significant parasite in Australia in terms of animal and human health. However, very little is known about its virome. In this study, next-generation sequencing of I. holocyclus salivary glands yielded a full-length genome sequence which phylogenetically groups with viruses classified in the Iflaviridae family and shares 45% amino acid similarity with its closest relative Bole hyalomma asiaticum virus 1. The sequence of this virus, provisionally named Ixodes holocyclus iflavirus (IhIV) has been identified in tick populations from northern New South Wales and Queensland, Australia and represents the first virus sequence reported from I. holocyclus.

Recombination of polynucleotide sequences using random or defined primers

DOEpatents

Arnold, Frances H.; Shao, Zhixin; Affholter, Joseph A.; Zhao, Huimin H; Giver, Lorraine J.

2000-01-01

A method for in vitro mutagenesis and recombination of polynucleotide sequences based on polymerase-catalyzed extension of primer oligonucleotides is disclosed. The method involves priming template polynucleotide(s) with random-sequences or defined-sequence primers to generate a pool of short DNA fragments with a low level of point mutations. The DNA fragments are subjected to denaturization followed by annealing and further enzyme-catalyzed DNA polymerization. This procedure is repeated a sufficient number of times to produce full-length genes which comprise mutants of the original template polynucleotides. These genes can be further amplified by the polymerase chain reaction and cloned into a vector for expression of the encoded proteins.

The Internet of Skills: The use of 5th Generation Telecommunications, Haptics, and Artificial Intelligence in Robotic Surgery.

PubMed

Kim, Sandra Sy; Dohler, Mischa; Dasgupta, Prokar

2018-05-11

Minimally invasive surgery (MIS) is well established in many surgical applications, with an estimated surgical robot device markets anticipated to reach $20 billion by 2021 (1). It yields many known advantages over traditional open surgery, examples being smaller incision sites resulting in decreased blood loss and risk of infection, decreased length of hospital stay and time to return to work, and improved cosmesis (2).The field as a whole suffers from shortcomings today, and thus does not live up to its full potential. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Three-dimensional ophthalmic optical coherence tomography with a refraction correction algorithm

NASA Astrophysics Data System (ADS)

Zawadzki, Robert J.; Leisser, Christoph; Leitgeb, Rainer; Pircher, Michael; Fercher, Adolf F.

2003-10-01

We built an optical coherence tomography (OCT) system with a rapid scanning optical delay (RSOD) line, which allows probing full axial eye length. The system produces Three-dimensional (3D) data sets that are used to generate 3D tomograms of the model eye. The raw tomographic data were processed by an algorithm, which is based on Snell"s law to correct the interface positions. The Zernike polynomials representation of the interfaces allows quantitative wave aberration measurements. 3D images of our results are presented to illustrate the capabilities of the system and the algorithm performance. The system allows us to measure intra-ocular distances.

ECB deacylase mutants

DOEpatents

Arnold, Frances H.; Shao, Zhixin; Zhao, Huimin; Giver, Lorraine J.

2002-01-01

A method for in vitro mutagenesis and recombination of polynucleotide sequences based on polymerase-catalyzed extension of primer oligonucleotides is disclosed. The method involves priming template polynucleotide(s) with random-sequences or defined-sequence primers to generate a pool of short DNA fragments with a low level of point mutations. The DNA fragments are subjected to denaturization followed by annealing and further enzyme-catalyzed DNA polymerization. This procedure is repeated a sufficient number of times to produce full-length genes which comprise mutants of the original template polynucleotides. These genes can be further amplified by the polymerase chain reaction and cloned into a vector for expression of the encoded proteins.

Recombination of polynucleotide sequences using random or defined primers

DOEpatents

Arnold, Frances H.; Shao, Zhixin; Affholter, Joseph A.; Zhao, Huimin; Giver, Lorraine J.

2001-01-01

A method for in vitro mutagenesis and recombination of polynucleotide sequences based on polymerase-catalyzed extension of primer oligonucleotides is disclosed. The method involves priming template polynucleotide(s) with random-sequences or defined-sequence primers to generate a pool of short DNA fragments with a low level of point mutations. The DNA fragments are subjected to denaturization followed by annealing and further enzyme-catalyzed DNA polymerization. This procedure is repeated a sufficient number of times to produce full-length genes which comprise mutants of the original template polynucleotides. These genes can be further amplified by the polymerase chain reaction and cloned into a vector for expression of the encoded proteins.

Separating full-length protein from aggregating proteolytic products using filter flow-through purification.

PubMed

Churion, Kelly A; Rogers, Robert E; Bayless, Kayla J; Bondos, Sarah E

2016-12-01

Separation of full-length protein from proteolytic products is challenging, since the properties used to isolate the protein can also be present in proteolytic products. Many separation techniques risk non-specific protein adhesion and/or require a lot of time, enabling continued proteolysis and aggregation after lysis. We demonstrate that proteolytic products aggregate for two different proteins. As a result, full-length protein can be rapidly separated from these fragments by filter flow-through purification, resulting in a substantial protein purity enhancement. This rapid approach is likely to be useful for intrinsically disordered proteins, whose repetitive sequence composition and flexible nature can facilitate aggregation. Copyright © 2016 Elsevier Inc. All rights reserved.

Loss of GATA-1 Full Length as a Cause of Diamond–Blackfan Anemia Phenotype

PubMed Central

Parrella, Sara; Aspesi, Anna; Quarello, Paola; Garelli, Emanuela; Pavesi, Elisa; Carando, Adriana; Nardi, Margherita; Ellis, Steven R.; Ramenghi, Ugo; Dianzani, Irma

2015-01-01

Mutations in the hematopoietic transcription factor GATA-1 alter the proliferation/differentiation of hemopoietic progenitors. Mutations in exon 2 interfere with the synthesis of the full-length isoform of GATA-1 and lead to the production of a shortened isoform, GATA-1s. These mutations have been found in patients with Diamond–Blackfan anemia (DBA), a congenital erythroid aplasia typically caused by mutations in genes encoding ribosomal proteins. We sequenced GATA-1 in 23 patients that were negative for mutations in the most frequently mutated DBA genes. One patient showed a c.2T > C mutation in the initiation codon leading to the loss of the full-length GATA-1 isoform. PMID:24453067

First full-length genome sequence of the polerovirus luffa aphid-borne yellows virus (LABYV) reveals the presence of at least two consensus sequences in an isolate from Thailand.

PubMed

Knierim, Dennis; Maiss, Edgar; Kenyon, Lawrence; Winter, Stephan; Menzel, Wulf

2015-10-01

Luffa aphid-borne yellows virus (LABYV) was proposed as the name for a previously undescribed polerovirus based on partial genome sequences obtained from samples of cucurbit plants collected in Thailand between 2008 and 2013. In this study, we determined the first full-length genome sequence of LABYV. Based on phylogenetic analysis and genome properties, it is clear that this virus represents a distinct species in the genus Polerovirus. Analysis of sequences from sample TH24, which was collected in 2010 from a luffa plant in Thailand, reveals the presence of two different full-length genome consensus sequences.

Extremely High Peak Power Obtained at 29 GHZ Microwave Pulse Generation

NASA Astrophysics Data System (ADS)

Rostov, V. V.; Gunin, A. V.; Romanchenko, I. V.; Pedos, M. S.; Rukin, S. N.; Sharypov, K. A.; Shunailov, S. A.; Ul'maskulov, M. R.; Yalandin, M. I.

2017-12-01

The paper presents research results on enhancing the peak power of microwave pulses with sub- and nanosecond length using a backward-wave oscillator (BWO) operating at 29 GHz frequency and possessing a reproducible phase structure. Experiments are conducted in two modes on a high-current electron accelerator with the required electron beam power. In the first (superradiation) mode, which utilizes the elongated slow-wave structure, the BWO peak power is 3 GW at 180 ns pulse duration (full width at halfmaximum, FWHM). In the second (quasi-stationary) mode, the BWO peak power reaches 600 MW at 2 ns pulse duration (FWHM). The phase spread from pulse to pulse can vary from units to several tens of percent in a nanosecond pulse mode. The experiments do not show any influence of microwave breakdown on the BWO power generation and radiation pulse duration.

Passive mode-locking of 3.25μm GaSb-based type-I quantum-well cascade diode lasers

NASA Astrophysics Data System (ADS)

Feng, Tao; Shterengas, Leon; Kipshidze, Gela; Hosoda, Takashi; Wang, Meng; Belenky, Gregory

2018-02-01

Passively mode-locked type-I quantum well cascade diode lasers emitting in the methane absorption band near 3.25 μm were designed, fabricated and characterized. The deep etched 5.5-μm-wide single spatial mode ridge waveguide design utilizing split-contact architecture was implemented. The devices with absorber to gain section length ratios of 11% and 5.5% were studied. Lasers with the longer absorber section ( 300 μm) generated smooth bell-shape-like emission spectrum with about 30 lasing modes at full-width-at-half-maximum level. Devices with reverse biased absorber section demonstrated stable radio frequency beat with nearly perfect Lorentzian shape over four orders of magnitude of intensity. The estimated pulse-to-pulse timing jitter was about 110 fs/cycle. Laser generated average power of more than 1 mW in mode-locked regime.

Identification of a new HIV-1 circulating recombinant form CRF65_cpx strain in Jilin, China.

PubMed

Wang, Jia-Ye; Chen, Xiao-Hong; Shao, Bing; Huo, Qing-Qing; Liu, Si-Yu; Li, Jin; Wang, Fu-Xiang

2018-05-04

This study reported a new HIV-1 circulating recombinant form CRF65_cpx virus isolated from a man who had sex with men (MSM) in Jilin, China. The near full-length genome of this virus was composed of fourteen mosaic gene fragments derived from CRF01_AE, subtype B' (Thai B) and subtype C, highly similar to the CRF65_cpx viruses recently identified in Yunnan and Anhui of China. Phylogenetic tree analysis suggested that this CRF65_cpx strain was not generated among MSM in Jilin, but originated in southern regions of China and spread to Jilin by MSM population. The emergence of CRF65_cpx in Jilin indicated HIV-1 epidemic in this area was more and more complicated and the MSM population has become the important source for generation of new recombinant viruses. Real-time surveillance of new HIV-1 infections among MSM population is quite required.

The design and synthesis of new synthetic low molecular weight heparins

PubMed Central

Chandarajoti, K.; Liu, J.; Pawlinski, R.

2016-01-01

Low molecular weight heparins (LMWH) have remained the most favorable form of heparin in clinics since 1990s’ owing to its predictable pharmacokinetic properties. However, LMWH is mainly eliminated through kidney, thus limits its use in renal-impaired patients. In addition, the anticoagulant activity of LMWH is only partially neutralized by protamine. LMWH is obtained from a full-length, highly sulfated polysaccharide harvested from porcine mucosal tissue. The depolymerization involved in LMWH production generates a broad size distribution of LMWH fragments (6-22 sugar residues). This, combined with the various methods used to produce commercial LMWHs, result in variable pharmacological and pharmacokinetic properties. An alternative, chemoenzymatic approach offers a method for the synthesis of LMWH that has the potential to overcome the limitations of current LMWHs. This review summarizes the application of a chemoenzymatic approach to generate LMWH and the rationale for development of a synthetic LMWH. PMID:26990516

The design and synthesis of new synthetic low-molecular-weight heparins.

PubMed

Chandarajoti, K; Liu, J; Pawlinski, R

2016-06-01

Low-molecular-weight heparin (LMWH) has remained the most favorable form of heparin in clinics since the 1990s owing to its predictable pharmacokinetic properties. However, LMWH is mainly eliminated through the kidney, which limits its use in renal-impaired patients. In addition, the anticoagulant activity of LMWH is only partially neutralized by protamine. LMWH is obtained from a full-length, highly sulfated polysaccharide harvested from porcine mucosal tissue. The depolymerization involved in LMWH production generates a broad distribution of LMWH fragments (6-22 sugar residues). This, combined with the various methods used to produce commercial LMWHs, results in variable pharmacological and pharmacokinetic properties. An alternative chemoenzymatic approach offers a method for the synthesis of LMWH that has the potential to overcome the limitations of current LMWHs. This review summarizes the application of a chemoenzymatic approach to generate LMWH and the rationale for development of a synthetic LMWH. © 2016 International Society on Thrombosis and Haemostasis.

Analysis of Tangential Slot Blowing on F/A-18 Isolated Forebody

NASA Technical Reports Server (NTRS)

Gee, Ken; Rizk, Yehia M.; Schiff, Lewis B.

1995-01-01

The generation of significant side forces and yawing moments on an F/A-18 fuselage through tangential slot blowing is analyzed using computational fluid dynamics. The effects of freestream Mach number, jet exit conditions, jet length, and jet location are studied. The effects of over- and underblowing on force and moment production are analyzed. Non-time-accurate solutions are obtained to determine the steady-state side forces, yawing moments, and surface pressure distributions generated by tangential slot blowing. Time-accurate solutions are obtained to study the force onset time lag of tangential slot blowing. Comparison with available experimental data from full-scale wind-tunnel and subscale wind-tunnel tests are made. This computational analysis complements the experimental results and provides a detailed understanding of the effects of tangential slot blowing on the flowfield about the isolated F/A-18 forebody. Additionally, it extends the slot-blowing database to transonic maneuvering Mach numbers.

Characterization of antigenic determinants in ApxIIA exotoxin capable of inducing protective immunity to Actinobacillus pleuropneumoniae challenge.

PubMed

Seo, Ki-Weon; Kim, Dong-Heon; Kim, Ah Hyun; Yoo, Han-Sang; Lee, Kyung-Yeol; Jang, Yong-Suk

2011-01-01

Actinobacillus pleuropneumoniae is the causative agent of porcine pleuropneumonia. Among the virulence factors of the pathogen, ApxIIA, a bacterial exotoxin, is expressed by many serotypes and presents a plausible target for vaccine development. We characterized the region within ApxIIA that induces a protective immune response against bacterial infection using mouse challenge model. Recombinant proteins spanning the length of ApxIIA were produced and antiserum to the full-length ApxIIA was induced in mice. This antiserum recognized fragments #2, #3 and #5 with high binding specificity, but showed poor recognition for fragments #1 and #4. Of the antisera induced in mice by injection of each fragments, only the antiserum to fragment #4 failed to efficiently recognize the full-length antigen, although the individual antisera recognized their cognate antigens with almost equal efficiency. The protective potency of the immunogenic proteins against a challenge injection of bacteria in vivo correlated well with the antibody titer. Fragment #5 induced the highest level of protective activity, comparable to that by the full-length protein. These results support the use of fragment #5 to produce a vaccine against A. pleuropneumoniae challenge, since the small antigen peptide is easier to handle than is the full-length protein and can be expressed efficiently in heterologous expression systems.

Assessment and optimization of theileria parva sporozoite full-length p67 antigen expression in mammalian cells

USDA-ARS?s Scientific Manuscript database

Delivery of various forms of recombinant Theileria parva sporozoite antigen (p67) has been shown to elicit antibody responses in cattle capable of providing protection against East Coast fever, the clinical disease caused by T. parva. Previous formulations of full-length and shorter recombinant vers...

Tula hantavirus isolate with the full-length ORF for nonstructural protein NSs survives for more consequent passages in interferon-competent cells than the isolate having truncated NSs ORF.

PubMed

Jääskeläinen, Kirsi M; Plyusnina, Angelina; Lundkvist, Ake; Vaheri, Antti; Plyusnin, Alexander

2008-01-11

The competitiveness of two Tula hantavirus (TULV) isolates, TULV/Lodz and TULV/Moravia, was evaluated in interferon (IFN) -competent and IFN-deficient cells. The two isolates differ in the length of the open reading frame (ORF) encoding the nonstructural protein NSs, which has previously been shown to inhibit IFN response in infected cells. In IFN-deficient Vero E6 cells both TULV isolates survived equally well. In contrast, in IFN-competent MRC5 cells TULV/Lodz isolate, that possesses the NSs ORF for the full-length protein of 90 aa, survived for more consequent passages than TULV/Moravia isolate, which contains the ORF for truncated NSs protein (66-67 aa). Our data show that expression of a full-length NSs protein is beneficial for the virus survival and competitiveness in IFN-competent cells and not essential in IFN-deficient cells. These results suggest that the N-terminal aa residues are important for the full activity of the NSs protein.

An improved and validated RNA HLA class I SBT approach for obtaining full length coding sequences.

PubMed

Gerritsen, K E H; Olieslagers, T I; Groeneweg, M; Voorter, C E M; Tilanus, M G J

2014-11-01

The functional relevance of human leukocyte antigen (HLA) class I allele polymorphism beyond exons 2 and 3 is difficult to address because more than 70% of the HLA class I alleles are defined by exons 2 and 3 sequences only. For routine application on clinical samples we improved and validated the HLA sequence-based typing (SBT) approach based on RNA templates, using either a single locus-specific or two overlapping group-specific polymerase chain reaction (PCR) amplifications, with three forward and three reverse sequencing reactions for full length sequencing. Locus-specific HLA typing with RNA SBT of a reference panel, representing the major antigen groups, showed identical results compared to DNA SBT typing. Alleles encountered with unknown exons in the IMGT/HLA database and three samples, two with Null and one with a Low expressed allele, have been addressed by the group-specific RNA SBT approach to obtain full length coding sequences. This RNA SBT approach has proven its value in our routine full length definition of alleles. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Single-cell full-length total RNA sequencing uncovers dynamics of recursive splicing and enhancer RNAs.

PubMed

Hayashi, Tetsutaro; Ozaki, Haruka; Sasagawa, Yohei; Umeda, Mana; Danno, Hiroki; Nikaido, Itoshi

2018-02-12

Total RNA sequencing has been used to reveal poly(A) and non-poly(A) RNA expression, RNA processing and enhancer activity. To date, no method for full-length total RNA sequencing of single cells has been developed despite the potential of this technology for single-cell biology. Here we describe random displacement amplification sequencing (RamDA-seq), the first full-length total RNA-sequencing method for single cells. Compared with other methods, RamDA-seq shows high sensitivity to non-poly(A) RNA and near-complete full-length transcript coverage. Using RamDA-seq with differentiation time course samples of mouse embryonic stem cells, we reveal hundreds of dynamically regulated non-poly(A) transcripts, including histone transcripts and long noncoding RNA Neat1. Moreover, RamDA-seq profiles recursive splicing in >300-kb introns. RamDA-seq also detects enhancer RNAs and their cell type-specific activity in single cells. Taken together, we demonstrate that RamDA-seq could help investigate the dynamics of gene expression, RNA-processing events and transcriptional regulation in single cells.

System for monitoring an industrial or biological process

DOEpatents

Gross, Kenneth C.; Wegerich, Stephan W.; Vilim, Rick B.; White, Andrew M.

1998-01-01

A method and apparatus for monitoring and responding to conditions of an industrial process. Industrial process signals, such as repetitive manufacturing, testing and operational machine signals, are generated by a system. Sensor signals characteristic of the process are generated over a time length and compared to reference signals over the time length. The industrial signals are adjusted over the time length relative to the reference signals, the phase shift of the industrial signals is optimized to the reference signals and the resulting signals output for analysis by systems such as SPRT.

System for monitoring an industrial or biological process

DOEpatents

Gross, K.C.; Wegerich, S.W.; Vilim, R.B.; White, A.M.

1998-06-30

A method and apparatus are disclosed for monitoring and responding to conditions of an industrial process. Industrial process signals, such as repetitive manufacturing, testing and operational machine signals, are generated by a system. Sensor signals characteristic of the process are generated over a time length and compared to reference signals over the time length. The industrial signals are adjusted over the time length relative to the reference signals, the phase shift of the industrial signals is optimized to the reference signals and the resulting signals output for analysis by systems such as SPRT. 49 figs.

[Sequencing and analysis of complete genome of rabies viruses isolated from Chinese Ferret-Badger and dog in Zhejiang province].

PubMed

Lei, Yong-Liang; Wang, Xiao-Guang; Tao, Xiao-Yan; Li, Hao; Meng, Sheng-Li; Chen, Xiu-Ying; Liu, Fu-Ming; Ye, Bi-Feng; Tang, Qing

2010-01-01

Based on sequencing the full-length genomes of four Chinese Ferret-Badger and dog, we analyze the properties of rabies viruses genetic variation in molecular level, get the information about rabies viruses prevalence and variation in Zhejiang, and enrich the genome database of rabies viruses street strains isolated from China. Rabies viruses in suckling mice were isolated, overlapped fragments were amplified by RT-PCR and full-length genomes were assembled to analyze the nucleotide and deduced protein similarities and phylogenetic analyses from Chinese Ferret-Badger, dog, sika deer, vole, used vaccine strain were determined. The four full-length genomes were sequenced completely and had the same genetic structure with the length of 11, 923 nts or 11, 925 nts including 58 nts-Leader, 1353 nts-NP, 894 nts-PP, 609 nts-MP, 1575 nts-GP, 6386 nts-LP, and 2, 5, 5 nts- intergenic regions(IGRs), 423 nts-Pseudogene-like sequence (psi), 70 nts-Trailer. The four full-length genomes were in accordance with the properties of Rhabdoviridae Lyssa virus by BLAST and multi-sequence alignment. The nucleotide and amino acid sequences among Chinese strains had the highest similarity, especially among animals of the same species. Of the four full-length genomes, the similarity in amino acid level was dramatically higher than that in nucleotide level, so the nucleotide mutations happened in these four genomes were most synonymous mutations. Compared with the reference rabies viruses, the lengths of the five protein coding regions had no change, no recombination, only with a few point mutations. It was evident that the five proteins appeared to be stable. The variation sites and types of the four genomes were similar to the reference vaccine or street strains. And the four strains were genotype 1 according to the multi-sequence and phylogenetic analyses, which possessed the distinct district characteristics of China. Therefore, these four rabies viruses are likely to be street viruses already existing in the natural world.

piggyBac transposons expressing full-length human dystrophin enable genetic correction of dystrophic mesoangioblasts.

PubMed

Loperfido, Mariana; Jarmin, Susan; Dastidar, Sumitava; Di Matteo, Mario; Perini, Ilaria; Moore, Marc; Nair, Nisha; Samara-Kuko, Ermira; Athanasopoulos, Takis; Tedesco, Francesco Saverio; Dickson, George; Sampaolesi, Maurilio; VandenDriessche, Thierry; Chuah, Marinee K

2016-01-29

Duchenne muscular dystrophy (DMD) is a genetic neuromuscular disorder caused by the absence of dystrophin. We developed a novel gene therapy approach based on the use of the piggyBac (PB) transposon system to deliver the coding DNA sequence (CDS) of either full-length human dystrophin (DYS: 11.1 kb) or truncated microdystrophins (MD1: 3.6 kb; MD2: 4 kb). PB transposons encoding microdystrophins were transfected in C2C12 myoblasts, yielding 65±2% MD1 and 66±2% MD2 expression in differentiated multinucleated myotubes. A hyperactive PB (hyPB) transposase was then deployed to enable transposition of the large-size PB transposon (17 kb) encoding the full-length DYS and green fluorescence protein (GFP). Stable GFP expression attaining 78±3% could be achieved in the C2C12 myoblasts that had undergone transposition. Western blot analysis demonstrated expression of the full-length human DYS protein in myotubes. Subsequently, dystrophic mesoangioblasts from a Golden Retriever muscular dystrophy dog were transfected with the large-size PB transposon resulting in 50±5% GFP-expressing cells after stable transposition. This was consistent with correction of the differentiated dystrophic mesoangioblasts following expression of full-length human DYS. These results pave the way toward a novel non-viral gene therapy approach for DMD using PB transposons underscoring their potential to deliver large therapeutic genes. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Salmo salar and Esox lucius full-length cDNA sequences reveal changes in evolutionary pressures on a post-tetraploidization genome

PubMed Central

2010-01-01

Background Salmonids are one of the most intensely studied fish, in part due to their economic and environmental importance, and in part due to a recent whole genome duplication in the common ancestor of salmonids. This duplication greatly impacts species diversification, functional specialization, and adaptation. Extensive new genomic resources have recently become available for Atlantic salmon (Salmo salar), but documentation of allelic versus duplicate reference genes remains a major uncertainty in the complete characterization of its genome and its evolution. Results From existing expressed sequence tag (EST) resources and three new full-length cDNA libraries, 9,057 reference quality full-length gene insert clones were identified for Atlantic salmon. A further 1,365 reference full-length clones were annotated from 29,221 northern pike (Esox lucius) ESTs. Pairwise dN/dS comparisons within each of 408 sets of duplicated salmon genes using northern pike as a diploid out-group show asymmetric relaxation of selection on salmon duplicates. Conclusions 9,057 full-length reference genes were characterized in S. salar and can be used to identify alleles and gene family members. Comparisons of duplicated genes show that while purifying selection is the predominant force acting on both duplicates, consistent with retention of functionality in both copies, some relaxation of pressure on gene duplicates can be identified. In addition, there is evidence that evolution has acted asymmetrically on paralogs, allowing one of the pair to diverge at a faster rate. PMID:20433749

Identification of a Novel LXXLL Motif in α-Actinin 4-spliced Isoform That Is Critical for Its Interaction with Estrogen Receptor α and Co-activators*

PubMed Central

Khurana, Simran; Chakraborty, Sharmistha; Zhao, Xuan; Liu, Yu; Guan, Dongyin; Lam, Minh; Huang, Wei; Yang, Sichun; Kao, Hung-Ying

2012-01-01

α-Actinins (ACTNs) are a family of proteins cross-linking actin filaments that maintain cytoskeletal organization and cell motility. Recently, it has also become clear that ACTN4 can function in the nucleus. In this report, we found that ACTN4 (full length) and its spliced isoform ACTN4 (Iso) possess an unusual LXXLL nuclear receptor interacting motif. Both ACTN4 (full length) and ACTN4 (Iso) potentiate basal transcription activity and directly interact with estrogen receptor α, although ACTN4 (Iso) binds ERα more strongly. We have also found that both ACTN4 (full length) and ACTN4 (Iso) interact with the ligand-independent and the ligand-dependent activation domains of estrogen receptor α. Although ACTN4 (Iso) interacts efficiently with transcriptional co-activators such as p300/CBP-associated factor (PCAF) and steroid receptor co-activator 1 (SRC-1), the full length ACTN4 protein either does not or does so weakly. More importantly, the flanking sequences of the LXXLL motif are important not only for interacting with nuclear receptors but also for the association with co-activators. Taken together, we have identified a novel extended LXXLL motif that is critical for interactions with both receptors and co-activators. This motif functions more efficiently in a spliced isoform of ACTN4 than it does in the full-length protein. PMID:22908231

Octave-spanning supercontinuum generation at telecommunications wavelengths in a precisely dispersion- and length-controlled silicon-wire waveguide with a double taper structure

NASA Astrophysics Data System (ADS)

Ishizawa, Atsushi; Goto, Takahiro; Kou, Rai; Tsuchizawa, Tai; Matsuda, Nobuyuki; Hitachi, Kenichi; Nishikawa, Tadashi; Yamada, Koji; Sogawa, Tetsuomi; Gotoh, Hideki

2017-07-01

We demonstrate on-chip octave-spanning supercontinuum (SC) generation with a Si-wire waveguide (SWG). We precisely controlled the SWG width so that the group velocity becomes flat over a wide wavelength range. By adjusting the SWG length, we could reduce the optical losses due to two-photon absorption and pulse propagation. In addition, for efficient coupling between the laser pulse and waveguide, we fabricated a two-step inverse taper at both ends of the SWG. Using a 600-nm-wide SWG, we were able to generate a broadband SC spectrum at wavelengths from 1060 to 2200 nm at a -40 dB level with only 50-pJ laser energy from an Er-doped fiber laser oscillator. We found that we can generate an on-chip broadband SC spectrum with an SWG with a length even as small as 1.7 mm.

Pulp regeneration in a full-length human tooth root using a hierarchical nanofibrous microsphere system.

PubMed

Li, Xiangwei; Ma, Chi; Xie, Xiaohua; Sun, Hongchen; Liu, Xiaohua

2016-04-15

While pulp regeneration using tissue engineering strategy has been explored for over a decade, successful regeneration of pulp tissues in a full-length human root with a one-end seal that truly simulates clinical endodontic treatment has not been achieved. To address this challenge, we designed and synthesized a unique hierarchical growth factor-loaded nanofibrous microsphere scaffolding system. In this system, vascular endothelial growth factor (VEGF) binds with heparin and is encapsulated in heparin-conjugated gelatin nanospheres, which are further immobilized in the nanofibers of an injectable poly(l-lactic acid) (PLLA) microsphere. This hierarchical microsphere system not only protects the VEGF from denaturation and degradation, but also provides excellent control of its sustained release. In addition, the nanofibrous PLLA microsphere integrates the extracellular matrix-mimicking architecture with a highly porous injectable form, efficiently accommodating dental pulp stem cells (DPSCs) and supporting their proliferation and pulp tissue formation. Our in vivo study showed the successful regeneration of pulp-like tissues that fulfilled the entire apical and middle thirds and reached the coronal third of the full-length root canal. In addition, a large number of blood vessels were regenerated throughout the canal. For the first time, our work demonstrates the success of pulp tissue regeneration in a full-length root canal, making it a significant step toward regenerative endodontics. The regeneration of pulp tissues in a full-length tooth root canal has been one of the greatest challenges in the field of regenerative endodontics, and one of the biggest barriers for its clinical application. In this study, we developed a unique approach to tackle this challenge, and for the first time, we successfully regenerated living pulp tissues in a full-length root canal, making it a significant step toward regenerative endodontics. This study will make positive scientific impact and interest the broad and multidisciplinary readership in the dental biomaterials and craniofacial tissue engineering community. Copyright © 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Reversal of a full-length mutant huntingtin neuronal cell phenotype by chemical inhibitors of polyglutamine-mediated aggregation

PubMed Central

Wang, Jin; Gines, Silvia; MacDonald, Marcy E; Gusella, James F

2005-01-01

Background Huntington's disease (HD) is an inherited neurodegenerative disorder triggered by an expanded polyglutamine tract in huntingtin that is thought to confer a new conformational property on this large protein. The propensity of small amino-terminal fragments with mutant, but not wild-type, glutamine tracts to self-aggregate is consistent with an altered conformation but such fragments occur relatively late in the disease process in human patients and mouse models expressing full-length mutant protein. This suggests that the altered conformational property may act within the full-length mutant huntingtin to initially trigger pathogenesis. Indeed, genotype-phenotype studies in HD have defined genetic criteria for the disease initiating mechanism, and these are all fulfilled by phenotypes associated with expression of full-length mutant huntingtin, but not amino-terminal fragment, in mouse models. As the in vitro aggregation of amino-terminal mutant huntingtin fragment offers a ready assay to identify small compounds that interfere with the conformation of the polyglutamine tract, we have identified a number of aggregation inhibitors, and tested whether these are also capable of reversing a phenotype caused by endogenous expression of mutant huntingtin in a striatal cell line from the HdhQ111/Q111 knock-in mouse. Results We screened the NINDS Custom Collection of 1,040 FDA approved drugs and bioactive compounds for their ability to prevent in vitro aggregation of Q58-htn 1–171 amino terminal fragment. Ten compounds were identified that inhibited aggregation with IC50 < 15 μM, including gossypol, gambogic acid, juglone, celastrol, sanguinarine and anthralin. Of these, both juglone and celastrol were effective in reversing the abnormal cellular localization of full-length mutant huntingtin observed in mutant HdhQ111/Q111 striatal cells. Conclusions At least some compounds identified as aggregation inhibitors also prevent a neuronal cellular phenotype caused by full-length mutant huntingtin, suggesting that in vitro fragment aggregation can act as a proxy for monitoring the disease-producing conformational property in HD. Thus, identification and testing of compounds that alter in vitro aggregation is a viable approach for defining potential therapeutic compounds that may act on the deleterious conformational property of full-length mutant huntingtin. PMID:15649316

Distinct spatiotemporal accumulation of N-truncated and full-length amyloid-β42 in Alzheimer's disease.

PubMed

Shinohara, Mitsuru; Koga, Shunsuke; Konno, Takuya; Nix, Jeremy; Shinohara, Motoko; Aoki, Naoya; Das, Pritam; Parisi, Joseph E; Petersen, Ronald C; Rosenberry, Terrone L; Dickson, Dennis W; Bu, Guojun

2017-12-01

Accumulation of amyloid-β peptides is a dominant feature in the pathogenesis of Alzheimer's disease; however, it is not clear how individual amyloid-β species accumulate and affect other neuropathological and clinical features in the disease. Thus, we compared the accumulation of N-terminally truncated amyloid-β and full-length amyloid-β, depending on disease stage as well as brain area, and determined how these amyloid-β species respectively correlate with clinicopathological features of Alzheimer's disease. To this end, the amounts of amyloid-β species and other proteins related to amyloid-β metabolism or Alzheimer's disease were quantified by enzyme-linked immunosorbent assays (ELISA) or theoretically calculated in 12 brain regions, including neocortical, limbic and subcortical areas from Alzheimer's disease cases (n = 19), neurologically normal elderly without amyloid-β accumulation (normal ageing, n = 13), and neurologically normal elderly with cortical amyloid-β accumulation (pathological ageing, n = 15). We observed that N-terminally truncated amyloid-β42 and full-length amyloid-β42 accumulations distributed differently across disease stages and brain areas, while N-terminally truncated amyloid-β40 and full-length amyloid-β40 accumulation showed an almost identical distribution pattern. Cortical N-terminally truncated amyloid-β42 accumulation was increased in Alzheimer's disease compared to pathological ageing, whereas cortical full-length amyloid-β42 accumulation was comparable between Alzheimer's disease and pathological ageing. Moreover, N-terminally truncated amyloid-β42 were more likely to accumulate more in specific brain areas, especially some limbic areas, while full-length amyloid-β42 tended to accumulate more in several neocortical areas, including frontal cortices. Immunoprecipitation followed by mass spectrometry analysis showed that several N-terminally truncated amyloid-β42 species, represented by pyroglutamylated amyloid-β11-42, were enriched in these areas, consistent with ELISA results. N-terminally truncated amyloid-β42 accumulation showed significant regional association with BACE1 and neprilysin, but not PSD95 that regionally associated with full-length amyloid-β42 accumulation. Interestingly, accumulations of tau and to a greater extent apolipoprotein E (apoE, encoded by APOE) were more strongly correlated with N-terminally truncated amyloid-β42 accumulation than those of other amyloid-β species across brain areas and disease stages. Consistently, immunohistochemical staining and in vitro binding assays showed that apoE co-localized and bound more strongly with pyroglutamylated amyloid-β11-x fibrils than full-length amyloid-β fibrils. Retrospective review of clinical records showed that accumulation of N-terminally truncated amyloid-β42 in cortical areas was associated with disease onset, duration and cognitive scores. Collectively, N-terminally truncated amyloid-β42 species have spatiotemporal accumulation patterns distinct from full-length amyloid-β42, likely due to different mechanisms governing their accumulations in the brain. These truncated amyloid-β species could play critical roles in the disease by linking other clinicopathological features of Alzheimer's disease. © The Author (2017). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

A heuristic for suffix solutions

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bilgory, A.; Gajski, D.D.

1986-01-01

The suffix problem has appeared in solutions of recurrence systems for parallel and pipelined machines and more recently in the design of gate and silicon compilers. In this paper the authors present two algorithms. The first algorithm generates parallel suffix solutions with minimum cost for a given length, time delay, availability of initial values, and fanout. This algorithm generates a minimal solution for any length n and depth range log/sub 2/ N to N. The second algorithm reduces the size of the solutions generated by the first algorithm.

Fast Fiber-Coupled Imaging Devices

DOE Office of Scientific and Technical Information (OSTI.GOV)

Brockington, Samuel; Case, Andrew; Witherspoon, Franklin Douglas

HyperV Technologies Corp. has successfully designed, built and experimentally demonstrated a full scale 1024 pixel 100 MegaFrames/s fiber coupled camera with 12 or 14 bits, and record lengths of 32K frames, exceeding our original performance objectives. This high-pixel-count, fiber optically-coupled, imaging diagnostic can be used for investigating fast, bright plasma events. In Phase 1 of this effort, a 100 pixel fiber-coupled fast streak camera for imaging plasma jet profiles was constructed and successfully demonstrated. The resulting response from outside plasma physics researchers emphasized development of increased pixel performance as a higher priority over increasing pixel count. In this Phase 2more » effort, HyperV therefore focused on increasing the sample rate and bit-depth of the photodiode pixel designed in Phase 1, while still maintaining a long record length and holding the cost per channel to levels which allowed up to 1024 pixels to be constructed. Cost per channel was 53.31 dollars, very close to our original target of $50 per channel. The system consists of an imaging "camera head" coupled to a photodiode bank with an array of optical fibers. The output of these fast photodiodes is then digitized at 100 Megaframes per second and stored in record lengths of 32,768 samples with bit depths of 12 to 14 bits per pixel. Longer record lengths are possible with additional memory. A prototype imaging system with up to 1024 pixels was designed and constructed and used to successfully take movies of very fast moving plasma jets as a demonstration of the camera performance capabilities. Some faulty electrical components on the 64 circuit boards resulted in only 1008 functional channels out of 1024 on this first generation prototype system. We experimentally observed backlit high speed fan blades in initial camera testing and then followed that with full movies and streak images of free flowing high speed plasma jets (at 30-50 km/s). Jet structure and jet collisions onto metal pillars in the path of the plasma jets were recorded in a single shot. This new fast imaging system is an attractive alternative to conventional fast framing cameras for applications and experiments where imaging events using existing techniques are inefficient or impossible. The development of HyperV's new diagnostic was split into two tracks: a next generation camera track, in which HyperV built, tested, and demonstrated a prototype 1024 channel camera at its own facility, and a second plasma community beta test track, where selected plasma physics programs received small systems of a few test pixels to evaluate the expected performance of a full scale camera on their experiments. These evaluations were performed as part of an unfunded collaboration with researchers at Los Alamos National Laboratory and the University of California at Davis. Results from the prototype 1024-pixel camera are discussed, as well as results from the collaborations with test pixel system deployment sites.« less

The correlation structure of several popular pseudorandom number generators

NASA Technical Reports Server (NTRS)

Neuman, F.; Merrick, R.; Martin, C. F.

1973-01-01

One of the desirable properties of a pseudorandom number generator is that the sequence of numbers it generates should have very low autocorrelation for all shifts except for zero shift and those that are multiples of its cycle length. Due to the simple methods of constructing random numbers, the ideal is often not quite fulfilled. A simple method of examining any random generator for previously unsuspected regularities is discussed. Once they are discovered it is often easy to derive the mathematical relationships, which describe the mathematical relationships, which describe the regular behavior. As examples, it is shown that high correlation exists in mixed and multiplicative congruential random number generators and prime moduli Lehmer generators for shifts a fraction of their cycle lengths.

Generation of expressed sequence tags for discovery of genes responsible for floral traits of Chrysanthemum morifolium by next-generation sequencing technology.

PubMed

Sasaki, Katsutomo; Mitsuda, Nobutaka; Nashima, Kenji; Kishimoto, Kyutaro; Katayose, Yuichi; Kanamori, Hiroyuki; Ohmiya, Akemi

2017-09-04

Chrysanthemum morifolium is one of the most economically valuable ornamental plants worldwide. Chrysanthemum is an allohexaploid plant with a large genome that is commercially propagated by vegetative reproduction. New cultivars with different floral traits, such as color, morphology, and scent, have been generated mainly by classical cross-breeding and mutation breeding. However, only limited genetic resources and their genome information are available for the generation of new floral traits. To obtain useful information about molecular bases for floral traits of chrysanthemums, we read expressed sequence tags (ESTs) of chrysanthemums by high-throughput sequencing using the 454 pyrosequencing technology. We constructed normalized cDNA libraries, consisting of full-length, 3'-UTR, and 5'-UTR cDNAs derived from various tissues of chrysanthemums. These libraries produced a total number of 3,772,677 high-quality reads, which were assembled into 213,204 contigs. By comparing the data obtained with those of full genome-sequenced species, we confirmed that our chrysanthemum contig set contained the majority of all expressed genes, which was sufficient for further molecular analysis in chrysanthemums. We confirmed that our chrysanthemum EST set (contigs) contained a number of contigs that encoded transcription factors and enzymes involved in pigment and aroma compound metabolism that was comparable to that of other species. This information can serve as an informative resource for identifying genes involved in various biological processes in chrysanthemums. Moreover, the findings of our study will contribute to a better understanding of the floral characteristics of chrysanthemums including the myriad cultivars at the molecular level.

NICU management and outcomes of infants with trisomy 21 without major anomalies.

PubMed

McAndrew, Sarah; Acharya, Krishna; Nghiem-Rao, T Hang; Leuthner, Steven; Clark, Reese; Lagatta, Joanne

2018-05-25

To describe how trisomy 21 affects neonatal intensive care management and outcomes of full-term infants without congenital anomalies. Retrospective cohort of full-term infants without anomalies with and without trisomy 21 admitted to Pediatrix NICUs from 2005 to 2012. We compared diagnoses, management, length of stay, and discharge outcomes. In all, 4623 infants with trisomy 21 and 606 770 infants without trisomy 21 were identified. One-third of infants in the NICU with and without trisomy 21 were full term without major anomalies. Trisomy 21 infants had more respiratory distress, thrombocytopenia, feeding problems, and pulmonary hypertension. They received respiratory support for a longer period of time and had a longer length of stay. One-third of infants with trisomy 21 admitted to the NICU are full term without major anomalies. Common diagnoses and greater respiratory needs place infants with trisomy 21 at risk for longer length of stay.

Effects of complex life cycles on genetic diversity: cyclical parthenogenesis

PubMed Central

Rouger, R; Reichel, K; Malrieu, F; Masson, J P; Stoeckel, S

2016-01-01

Neutral patterns of population genetic diversity in species with complex life cycles are difficult to anticipate. Cyclical parthenogenesis (CP), in which organisms undergo several rounds of clonal reproduction followed by a sexual event, is one such life cycle. Many species, including crop pests (aphids), human parasites (trematodes) or models used in evolutionary science (Daphnia), are cyclical parthenogens. It is therefore crucial to understand the impact of such a life cycle on neutral genetic diversity. In this paper, we describe distributions of genetic diversity under conditions of CP with various clonal phase lengths. Using a Markov chain model of CP for a single locus and individual-based simulations for two loci, our analysis first demonstrates that strong departures from full sexuality are observed after only a few generations of clonality. The convergence towards predictions made under conditions of full clonality during the clonal phase depends on the balance between mutations and genetic drift. Second, the sexual event of CP usually resets the genetic diversity at a single locus towards predictions made under full sexuality. However, this single recombination event is insufficient to reshuffle gametic phases towards full-sexuality predictions. Finally, for similar levels of clonality, CP and acyclic partial clonality (wherein a fixed proportion of individuals are clonally produced within each generation) differentially affect the distribution of genetic diversity. Overall, this work provides solid predictions of neutral genetic diversity that may serve as a null model in detecting the action of common evolutionary or demographic processes in cyclical parthenogens (for example, selection or bottlenecks). PMID:27436524

Cloning of a coconut endosperm cDNA encoding a 1-acyl-sn-glycerol-3-phosphate acyltransferase that accepts medium-chain-length substrates.

PubMed Central

Knutzon, D S; Lardizabal, K D; Nelsen, J S; Bleibaum, J L; Davies, H M; Metz, J G

1995-01-01

Immature coconut (Cocos nucifera) endosperm contains a 1-acyl-sn-glycerol-3-phosphate acyltransferase (LPAAT) activity that shows a preference for medium-chain-length fatty acyl-coenzyme A substrates (H.M. Davies, D.J. Hawkins, J.S. Nelsen [1995] Phytochemistry 39:989-996). Beginning with solubilized membrane preparations, we have used chromatographic separations to identify a polypeptide with an apparent molecular mass of 29 kD, whose presence in various column fractions correlates with the acyltransferase activity detected in those same fractions. Amino acid sequence data obtained from several peptides generated from this protein were used to isolate a full-length clone from a coconut endosperm cDNA library. Clone pCGN5503 contains a 1325-bp cDNA insert with an open reading frame encoding a 308-amino acid protein with a calculated molecular mass of 34.8 kD. Comparison of the deduced amino acid sequence of pCGN5503 to sequences in the data banks revealed significant homology to other putative LPAAT sequences. Expression of the coconut cDNA in Escherichia coli conferred upon those cells a novel LPAAT activity whose substrate activity profile matched that of the coconut enzyme. PMID:8552723

Decreasing initial telomere length in humans intergenerationally understates age-associated telomere shortening

PubMed Central

Holohan, Brody; De Meyer, Tim; Batten, Kimberly; Mangino, Massimo; Hunt, Steven C; Bekaert, Sofie; De Buyzere, Marc L; Rietzschel, Ernst R; Spector, Tim D; Wright, Woodring E; Shay, Jerry W

2015-01-01

Telomere length shortens with aging, and short telomeres have been linked to a wide variety of pathologies. Previous studies suggested a discrepancy in age-associated telomere shortening rate estimated by cross-sectional studies versus the rate measured in longitudinal studies, indicating a potential bias in cross-sectional estimates. Intergenerational changes in initial telomere length, such as that predicted by the previously described effect of a father’s age at birth of his offspring (FAB), could explain the discrepancy in shortening rate measurements. We evaluated whether changes occur in initial telomere length over multiple generations in three large datasets and identified paternal birth year (PBY) as a variable that reconciles the difference between longitudinal and cross-sectional measurements. We also clarify the association between FAB and offspring telomere length, demonstrating that this effect is substantially larger than reported in the past. These results indicate the presence of a downward secular trend in telomere length at birth over generational time with potential public health implications. PMID:25952108

Influence of different heating types on the pumping performance of a bubble pump

NASA Astrophysics Data System (ADS)

Bierling, Bernd; Schmid, Fabian; Spindler, Klaus

2017-11-01

This study presents an experimental investigation of the influence of different heating types on the pumping performance of a bubble pump. A test rig was set up at the Institute of Thermodynamics and Thermal Engineering (ITW), University of Stuttgart. The vertical lift tube is made of copper with an inner diameter of 8 mm and a length of 1.91 m. The working fluid is demineralized water. The test rig offers the possibility to vary the supplied heat flow (0 W - 750 W), the resulting supplied heat flux and the location of the heating. Investigations were carried out using spot heating, partial-length heating and full-length heating. A Coriolis mass flowmeter was successfully implemented which measures the vapor mass flow rate continuously. The improvement of the vapor mass flow rate measurement by using the continuous measurement method compared to a discontinuous one is discussed. Furthermore, the influence of an unstable inlet temperature of the working fluid entering the lift tube on the pumping performance is investigated. The focus of this publication lies on the build-up of the test rig with the measurement setup and the analysis of the pumping performance for the three heating types. The measurement results show a big influence of the heating type on the pumping performance. The lower the relative length of the heating, the higher is the pumping ratio which is defined as the lifted liquid mass flow rate in relation to the generated vapor mass flow rate.

Mutation-Specific Effects on Thin Filament Length in Thin Filament Myopathy

PubMed Central

de Winter, Josine M.; Joureau, Barbara; Lee, Eun-Jeong; Kiss, Balázs; Yuen, Michaela; Gupta, Vandana A.; Pappas, Christopher T.; Gregorio, Carol C.; Stienen, Ger J. M.; Edvardson, Simon; Wallgren-Pettersson, Carina; Lehtokari, Vilma-Lotta; Pelin, Katarina; Malfatti, Edoardo; Romero, Norma B.; van Engelen, Baziel G.; Voermans, Nicol C.; Donkervoort, Sandra; Bönnemann, C. G.; Clarke, Nigel F.; Beggs, Alan H.; Granzier, Henk; Ottenheijm, Coen A. C.

2016-01-01

Objective Thin filament myopathies are among the most common nondystrophic congenital muscular disorders, and are caused by mutations in genes encoding proteins that are associated with the skeletal muscle thin filament. Mechanisms underlying muscle weakness are poorly understood, but might involve the length of the thin filament, an important determinant of force generation. Methods We investigated the sarcomere length-dependence of force, a functional assay that provides insights into the contractile strength of muscle fibers as well as the length of the thin filaments, in muscle fibers from 51 patients with thin filament myopathy caused by mutations in NEB, ACTA1, TPM2, TPM3, TNNT1, KBTBD13, KLHL40, and KLHL41. Results Lower force generation was observed in muscle fibers from patients of all genotypes. In a subset of patients who harbor mutations in NEB and ACTA1, the lower force was associated with downward shifted force–sarcomere length relations, indicative of shorter thin filaments. Confocal microscopy confirmed shorter thin filaments in muscle fibers of these patients. A conditional Neb knockout mouse model, which recapitulates thin filament myopathy, revealed a compensatory mechanism; the lower force generation that was associated with shorter thin filaments was compensated for by increasing the number of sarcomeres in series. This allowed muscle fibers to operate at a shorter sarcomere length and maintain optimal thin–thick filament overlap. Interpretation These findings might provide a novel direction for the development of therapeutic strategies for thin filament myopathy patients with shortened thin filament lengths. PMID:27074222

Inhibitors for Androgen Receptor Activation Surfaces

DTIC Science & Technology

2006-09-01

Thyroid hormone, 3,5,3′-triiodo-L-thyronine (T3; Sigma) P R O T O C O L www.stke.org/cgi/content/full/sigtrans;2006/341/pl3 Page 4 TNT T7 quick coupled...SRC2 full-length protein was obtained by using a TNT T7 quick coupled transcrip- tion/translation system. Transformation and preparation of bacterial...is conducted in triplicate for T3 and hit compounds. 1. Produce full-length hTRβ using a TNT T7 quick-coupled transcription translation system

Using RNA-Seq to assemble a rose transcriptome with more than 13,000 full-length expressed genes and to develop the WagRhSNP 68k Axiom SNP array for rose (Rosa L.).

PubMed

Koning-Boucoiran, Carole F S; Esselink, G Danny; Vukosavljev, Mirjana; van 't Westende, Wendy P C; Gitonga, Virginia W; Krens, Frans A; Voorrips, Roeland E; van de Weg, W Eric; Schulz, Dietmar; Debener, Thomas; Maliepaard, Chris; Arens, Paul; Smulders, Marinus J M

2015-01-01

In order to develop a versatile and large SNP array for rose, we set out to mine ESTs from diverse sets of rose germplasm. For this RNA-Seq libraries containing about 700 million reads were generated from tetraploid cut and garden roses using Illumina paired-end sequencing, and from diploid Rosa multiflora using 454 sequencing. Separate de novo assemblies were performed in order to identify single nucleotide polymorphisms (SNPs) within and between rose varieties. SNPs among tetraploid roses were selected for constructing a genotyping array that can be employed for genetic mapping and marker-trait association discovery in breeding programs based on tetraploid germplasm, both from cut roses and from garden roses. In total 68,893 SNPs were included on the WagRhSNP Axiom array. Next, an orthology-guided assembly was performed for the construction of a non-redundant rose transcriptome database. A total of 21,740 transcripts had significant hits with orthologous genes in the strawberry (Fragaria vesca L.) genome. Of these 13,390 appeared to contain the full-length coding regions. This newly established transcriptome resource adds considerably to the currently available sequence resources for the Rosaceae family in general and the genus Rosa in particular.

Both the stroma and thylakoid lumen of tobacco chloroplasts are competent for the formation of disulphide bonds in recombinant proteins.

PubMed

Bally, Julia; Paget, Eric; Droux, Michel; Job, Claudette; Job, Dominique; Dubald, Manuel

2008-01-01

Plant chloroplasts are promising vehicles for recombinant protein production, but the process of protein folding in these organelles is not well understood in comparison with that in prokaryotic systems, such as Escherichia coli. This is particularly true for disulphide bond formation which is crucial for the biological activity of many therapeutic proteins. We have investigated the capacity of tobacco (Nicotiana tabacum) chloroplasts to efficiently form disulphide bonds in proteins by expressing in this plant cell organelle a well-known bacterial enzyme, alkaline phosphatase, whose activity and stability strictly depend on the correct formation of two intramolecular disulphide bonds. Plastid transformants have been generated that express either the mature enzyme, localized in the stroma, or the full-length coding region, including its signal peptide. The latter has the potential to direct the recombinant alkaline phosphatase into the lumen of thylakoids, giving access to this even less well-characterized organellar compartment. We show that the chloroplast stroma supports the formation of an active enzyme, unlike a normal bacterial cytosol. Sorting of alkaline phosphatase to the thylakoid lumen occurs in the plastid transformants translating the full-length coding region, and leads to larger amounts and more active enzyme. These results are compared with those obtained in bacteria. The implications of these findings on protein folding properties and competency of chloroplasts for disulphide bond formation are discussed.

Novel Leptospira interrogans protein Lsa32 is expressed during infection and binds laminin and plasminogen.

PubMed

Domingos, Renan F; Fernandes, Luis G; Romero, Eliete C; de Morais, Zenaide M; Vasconcellos, Silvio A; Nascimento, Ana L T O

2015-04-01

Pathogenic Leptospira is the aetiological agent of leptospirosis, a life-threatening disease of human and veterinary concern. The quest for novel antigens that could mediate host-pathogen interactions is being pursued. Owing to their location, these antigens have the potential to elicit numerous activities, including immune response and adhesion. This study focuses on a hypothetical protein of Leptospira, encoded by the gene LIC11089, and its three derived fragments: the N-terminal, intermediate and C terminus regions. The gene coding for the full-length protein and fragments was cloned and expressed in Escherichia coli BL21(SI) strain by using the expression vector pAE. The recombinant protein and fragments tagged with hexahistidine at the N terminus were purified by metal affinity chromatography. The leptospiral full-length protein, named Lsa32 (leptospiral surface adhesin, 32 kDa), adheres to laminin, with the C terminus region being responsible for this interaction. Lsa32 binds to plasminogen in a dose-dependent fashion, generating plasmin when an activator is provided. Moreover, antibodies present in leptospirosis serum samples were able to recognize Lsa32. Lsa32 is most likely a new surface protein of Leptospira, as revealed by proteinase K susceptibility. Altogether, our data suggest that this multifaceted protein is expressed during infection and may play a role in host-L. interrogans interactions. © 2015 The Authors.

Functional endogenous LINE-1 retrotransposons are expressed and mobilized in rat chloroleukemia cells.

PubMed

Kirilyuk, Alexander; Tolstonog, Genrich V; Damert, Annette; Held, Ulrike; Hahn, Silvia; Löwer, Roswitha; Buschmann, Christian; Horn, Axel V; Traub, Peter; Schumann, Gerald G

2008-02-01

LINE-1 (L1) is a highly successful autonomous non-LTR retrotransposon and a major force shaping mammalian genomes. Although there are about 600 000 L1 copies covering 23% of the rat genome, full-length rat L1s (L1Rn) with intact open reading frames (ORFs) representing functional master copies for retrotransposition have not been identified yet. In conjunction with studies to elucidate the role of L1 retrotransposons in tumorigenesis, we isolated and characterized 10 different cDNAs from transcribed full-length L1Rn elements in rat chloroleukemia (RCL) cells, each encoding intact ORF1 proteins (ORF1p). We identified the first functional L1Rn retrotransposon from this pool of cDNAs, determined its activity in HeLa cells and in the RCL cell line the cDNAs originated from and demonstrate that it is mobilized in the tumor cell line in which it is expressed. Furthermore, we generated monoclonal antibodies directed against L1Rn ORF1 and ORF2-encoded recombinant proteins, analyzed the expression of L1-encoded proteins and found ORF1p predominantly in the nucleus. Our results support the hypothesis that the reported explosive amplification of genomic L1Rn sequences after their transcriptional activation in RCL cells is based on L1 retrotransposition. Therefore, L1 activity might be one cause for genomic instability observed during the progression of leukemia.

A full-length cDNA infectious clone of North American type 1 porcine reproductive and respiratory syndrome virus: expression of green fluorescent protein in the Nsp2 region.

PubMed

Fang, Ying; Rowland, Raymond R R; Roof, Michael; Lunney, Joan K; Christopher-Hennings, Jane; Nelson, Eric A

2006-12-01

The recent emergence of a unique group of North American type 1 porcine reproductive and respiratory syndrome virus (PRRSV) in the United States presents new disease control problems for a swine industry that has already been impacted seriously by North American type 2 PRRSV. In this study, a full-length cDNA infectious clone was generated from a low-virulence North American type 1 PRRSV isolate, SD01-08. In vitro studies demonstrated that the cloned virus maintained growth properties similar to those of the parental virus. Virological, pathological, and immunological observations from animals challenged with cloned viruses were similar to those from animals challenged with the parental virus and a modified live virus vaccine. To further explore the potential use as a viral backbone for expressing foreign genes, the green fluorescent protein (GFP) was inserted into a unique deletion site located at amino acid positions 348 and 349 of the predicted Nsp2 region in the virus, and expression of the Nsp2-GFP fusion protein was visualized by fluorescent microscopy. The availability of this North American type 1 infectious clone provides an important research tool for further study of the basic viral biology and pathogenic mechanisms of this group of type 1 PRRSV in the United States.

A Full-Length cDNA Infectious Clone of North American Type 1 Porcine Reproductive and Respiratory Syndrome Virus: Expression of Green Fluorescent Protein in the Nsp2 Region▿

PubMed Central

Fang, Ying; Rowland, Raymond R. R.; Roof, Michael; Lunney, Joan K.; Christopher-Hennings, Jane; Nelson, Eric A.

2006-01-01

The recent emergence of a unique group of North American type 1 porcine reproductive and respiratory syndrome virus (PRRSV) in the United States presents new disease control problems for a swine industry that has already been impacted seriously by North American type 2 PRRSV. In this study, a full-length cDNA infectious clone was generated from a low-virulence North American type 1 PRRSV isolate, SD01-08. In vitro studies demonstrated that the cloned virus maintained growth properties similar to those of the parental virus. Virological, pathological, and immunological observations from animals challenged with cloned viruses were similar to those from animals challenged with the parental virus and a modified live virus vaccine. To further explore the potential use as a viral backbone for expressing foreign genes, the green fluorescent protein (GFP) was inserted into a unique deletion site located at amino acid positions 348 and 349 of the predicted Nsp2 region in the virus, and expression of the Nsp2-GFP fusion protein was visualized by fluorescent microscopy. The availability of this North American type 1 infectious clone provides an important research tool for further study of the basic viral biology and pathogenic mechanisms of this group of type 1 PRRSV in the United States. PMID:16971421

Constructing high complexity synthetic libraries of long ORFs using in vitro selection

NASA Technical Reports Server (NTRS)

Cho, G.; Keefe, A. D.; Liu, R.; Wilson, D. S.; Szostak, J. W.

2000-01-01

We present a method that can significantly increase the complexity of protein libraries used for in vitro or in vivo protein selection experiments. Protein libraries are often encoded by chemically synthesized DNA, in which part of the open reading frame is randomized. There are, however, major obstacles associated with the chemical synthesis of long open reading frames, especially those containing random segments. Insertions and deletions that occur during chemical synthesis cause frameshifts, and stop codons in the random region will cause premature termination. These problems can together greatly reduce the number of full-length synthetic genes in the library. We describe a strategy in which smaller segments of the synthetic open reading frame are selected in vitro using mRNA display for the absence of frameshifts and stop codons. These smaller segments are then ligated together to form combinatorial libraries of long uninterrupted open reading frames. This process can increase the number of full-length open reading frames in libraries by up to two orders of magnitude, resulting in protein libraries with complexities of greater than 10(13). We have used this methodology to generate three types of displayed protein library: a completely random sequence library, a library of concatemerized oligopeptide cassettes with a propensity for forming amphipathic alpha-helical or beta-strand structures, and a library based on one of the most common enzymatic scaffolds, the alpha/beta (TIM) barrel. Copyright 2000 Academic Press.

Sequencing, Analysis, and Annotation of Expressed Sequence Tags for Camelus dromedarius

PubMed Central

Al-Swailem, Abdulaziz M.; Shehata, Maher M.; Abu-Duhier, Faisel M.; Al-Yamani, Essam J.; Al-Busadah, Khalid A.; Al-Arawi, Mohammed S.; Al-Khider, Ali Y.; Al-Muhaimeed, Abdullah N.; Al-Qahtani, Fahad H.; Manee, Manee M.; Al-Shomrani, Badr M.; Al-Qhtani, Saad M.; Al-Harthi, Amer S.; Akdemir, Kadir C.; Otu, Hasan H.

2010-01-01

Despite its economical, cultural, and biological importance, there has not been a large scale sequencing project to date for Camelus dromedarius. With the goal of sequencing complete DNA of the organism, we first established and sequenced camel EST libraries, generating 70,272 reads. Following trimming, chimera check, repeat masking, cluster and assembly, we obtained 23,602 putative gene sequences, out of which over 4,500 potentially novel or fast evolving gene sequences do not carry any homology to other available genomes. Functional annotation of sequences with similarities in nucleotide and protein databases has been obtained using Gene Ontology classification. Comparison to available full length cDNA sequences and Open Reading Frame (ORF) analysis of camel sequences that exhibit homology to known genes show more than 80% of the contigs with an ORF>300 bp and ∼40% hits extending to the start codons of full length cDNAs suggesting successful characterization of camel genes. Similarity analyses are done separately for different organisms including human, mouse, bovine, and rat. Accompanying web portal, CAGBASE (http://camel.kacst.edu.sa/), hosts a relational database containing annotated EST sequences and analysis tools with possibility to add sequences from public domain. We anticipate our results to provide a home base for genomic studies of camel and other comparative studies enabling a starting point for whole genome sequencing of the organism. PMID:20502665

Length-independent structural similarities enrich the antibody CDR canonical class model.

PubMed

Nowak, Jaroslaw; Baker, Terry; Georges, Guy; Kelm, Sebastian; Klostermann, Stefan; Shi, Jiye; Sridharan, Sudharsan; Deane, Charlotte M

2016-01-01

Complementarity-determining regions (CDRs) are antibody loops that make up the antigen binding site. Here, we show that all CDR types have structurally similar loops of different lengths. Based on these findings, we created length-independent canonical classes for the non-H3 CDRs. Our length variable structural clusters show strong sequence patterns suggesting either that they evolved from the same original structure or result from some form of convergence. We find that our length-independent method not only clusters a larger number of CDRs, but also predicts canonical class from sequence better than the standard length-dependent approach. To demonstrate the usefulness of our findings, we predicted cluster membership of CDR-L3 sequences from 3 next-generation sequencing datasets of the antibody repertoire (over 1,000,000 sequences). Using the length-independent clusters, we can structurally classify an additional 135,000 sequences, which represents a ∼20% improvement over the standard approach. This suggests that our length-independent canonical classes might be a highly prevalent feature of antibody space, and could substantially improve our ability to accurately predict the structure of novel CDRs identified by next-generation sequencing.

Two octaves spanning supercontinuum in highly nonlinear As2Se3 nanophotonic crystal fiber for midinfrared applications

NASA Astrophysics Data System (ADS)

Baili, Amira; Cherif, Rim; Zghal, Mourad

2015-01-01

A new design of all-normal and near-zero flattened dispersion based on chalcogenide nanophotonic crystal fiber (PCF) has been proposed to generate smooth and ultra-broadband supercontinuum (SC) in the midinfrared (IR) region. With the optimized geometric parameters, the As2Se3 nano-PCF has been found to be suitable for two-octave supercontinuum generation (SCG). We designed a nano-PCF having a flat top dispersion curve with a maximum value of -2.3 [ps/(nm km)] and a large nonlinear coefficient equal to 7250 W around the wavelength of 5.24 μm. By numerical simulations, we predict the generation of a very broadband SC in the mid-IR region extending from 2 to 10 μm in only 2-mm fiber lengths by using a femtosecond laser having a full-width at half-maximum of 50 fs and a relatively low energy of E=80 pJ. The generated SC demonstrates perfect coherence property over the entire bandwidth. SC generation extended into the mid-IR spectral region has potential usefulness in a variety of applications requiring a broad and mid-IR spectrum, such as WDM sources, fiber sensing, IR spectroscopy, fiber laser, and optical tomography coherence.

Pyroglutamate-Aβ 3 and 11 colocalize in amyloid plaques in Alzheimer's disease cerebral cortex with pyroglutamate-Aβ 11 forming the central core.

PubMed

Sullivan, Christopher P; Berg, Eric A; Elliott-Bryant, Rosemary; Fishman, Jordan B; McKee, Ann C; Morin, Peter J; Shia, Michael A; Fine, Richard E

2011-11-14

N-terminal truncated amyloid beta (Aβ) derivatives, especially the forms having pyroglutamate at the 3 position (AβpE3) or at the 11 position (AβpE11) have become the topic of considerable study. AβpE3 is known to make up a substantial portion of the Aβ species in senile plaques while AβpE11 has received less attention. We have generated very specific polyclonal antibodies against both species. Each antibody recognizes only the antigen against which it was generated on Western blots and neither recognizes full length Aβ. Both anti-AβpE3 and anti-AβpE11 stain senile plaques specifically in Alzheimer's disease cerebral cortex and colocalize with Aβ, as shown by confocal microscopy. In a majority of plaques examined, AβpE11 was observed to be the dominant form in the innermost core. These data suggest that AβpE11 may serve as a generating site for senile plaque formation. Published by Elsevier Ireland Ltd.

TSAR User’s Manual: A Program for Assessing the Effects of Conventional and Chemical Attacks on Sortie Generation. Volume 3. Variable and Array Definitions, and other Program Aids

DTIC Science & Technology

1990-09-01

array. LTHPER Length of the MPPERS array. LTHQPA Length of the QPA array. LTHXRT Length of the XROOT array. MAXACN Maximum number of aircraft that can...3 Time remaining until the ready-to-fly time at time of report Number of XROOT Array Entries (GENERATED) NROOT (MAXT) The total number of entries in...the XROOT array for each aircraft type. AIS Station Status NSTAT (NOSTAT, I, MAXB) I = 1 Total number of stations of each type on base = 2 Number in

Changes in average length of stay and average charges generated following institution of PSRO review.

PubMed Central

Westphal, M; Frazier, E; Miller, M C

1979-01-01

A five-year review of accounting data at a university hospital shows that immediately following institution of concurrent PSRO admission and length of stay review of Medicare-Medicaid patients, there was a significant decrease in length of stay and a fall in average charges generated per patient against the inflationary trend. Similar changes did not occur for the non-Medicare-Medicaid patients who were not reviewed. The observed changes occurred even though the review procedure rarely resulted in the denial of services to patients, suggesting an indirect effect of review. PMID:393658

Cost-effective sequencing of full-length cDNA clones powered by a de novo-reference hybrid assembly.

PubMed

Kuroshu, Reginaldo M; Watanabe, Junichi; Sugano, Sumio; Morishita, Shinichi; Suzuki, Yutaka; Kasahara, Masahiro

2010-05-07

Sequencing full-length cDNA clones is important to determine gene structures including alternative splice forms, and provides valuable resources for experimental analyses to reveal the biological functions of coded proteins. However, previous approaches for sequencing cDNA clones were expensive or time-consuming, and therefore, a fast and efficient sequencing approach was demanded. We developed a program, MuSICA 2, that assembles millions of short (36-nucleotide) reads collected from a single flow cell lane of Illumina Genome Analyzer to shotgun-sequence approximately 800 human full-length cDNA clones. MuSICA 2 performs a hybrid assembly in which an external de novo assembler is run first and the result is then improved by reference alignment of shotgun reads. We compared the MuSICA 2 assembly with 200 pooled full-length cDNA clones finished independently by the conventional primer-walking using Sanger sequencers. The exon-intron structure of the coding sequence was correct for more than 95% of the clones with coding sequence annotation when we excluded cDNA clones insufficiently represented in the shotgun library due to PCR failure (42 out of 200 clones excluded), and the nucleotide-level accuracy of coding sequences of those correct clones was over 99.99%. We also applied MuSICA 2 to full-length cDNA clones from Toxoplasma gondii, to confirm that its ability was competent even for non-human species. The entire sequencing and shotgun assembly takes less than 1 week and the consumables cost only approximately US$3 per clone, demonstrating a significant advantage over previous approaches.

A conserved truncated isoform of the ATR-X syndrome protein lacking the SWI/SNF-homology domain.

PubMed

Garrick, David; Samara, Vassiliki; McDowell, Tarra L; Smith, Andrew J H; Dobbie, Lorraine; Higgs, Douglas R; Gibbons, Richard J

2004-02-04

Mutations in the ATRX gene cause a severe X-linked mental retardation syndrome that is frequently associated with alpha thalassemia (ATR-X syndrome). The previously characterized ATRX protein (approximately 280 kDa) contains both a Plant homeodomain (PHD)-like zinc finger motif as well as an ATPase domain of the SNF2 family. These motifs suggest that ATRX may function as a regulator of gene expression, probably by exerting an effect on chromatin structure, although the exact cellular role of ATRX has not yet been fully elucidated. Here we characterize a truncated (approximately 200 kDa) isoform of ATRX (called here ATRXt) that has been highly conserved between mouse and human. In both species, ATRXt arises due to the failure to splice intron 11 from the primary transcript, and the use of a proximal intronic poly(A) signal. We show that the relative expression of the full length and ATRXt isoforms is subject to tissue-specific regulation. The ATRXt isoform contains the PHD-like domain but not the SWI/SNF-like motifs and is therefore unlikely to be functionally equivalent to the full length protein. We used indirect immunofluorescence to demonstrate that the full length and ATRXt isoforms are colocalized at blocks of pericentromeric heterochromatin but unlike full length ATRX, the truncated isoform does not associate with promyelocytic leukemia (PML) nuclear bodies. The high degree of conservation of ATRXt and the tight regulation of its expression relative to the full length protein suggest that this truncated isoform fulfills an important biological function.

A truncated Wnt7a retains full biological activity in skeletal muscle

NASA Astrophysics Data System (ADS)

von Maltzahn, Julia; Zinoviev, Radoslav; Chang, Natasha C.; Bentzinger, C. Florian; Rudnicki, Michael A.

2013-11-01

Wnt signaling has essential roles during embryonic development and tissue homoeostasis. Wnt proteins are post-translationally modified and the attachment of a palmitate moiety at two conserved residues is believed to be a prerequisite for the secretion and function of Wnt proteins. Here we demonstrate that a mammalian Wnt protein can be fully functional without palmitoylation. We generate a truncated Wnt7a variant, consisting of the C-terminal 137 amino acids lacking the conserved palmitoylation sites and show that it retains full biological activity in skeletal muscle. This includes binding to and signaling through its receptor Fzd7 to stimulate symmetric expansion of satellite stem cells by activating the planar-cell polarity pathway and inducing myofibre hypertrophy by signaling through the AKT/mTOR pathway. Furthermore, this truncated Wnt7a shows enhanced secretion and dispersion compared with the full-length protein. Together, these findings open important new avenues for the development of Wnt7a as a treatment for muscle-wasting diseases and have broad implications for the therapeutic use of Wnts as biologics.

Cloning, characterization, expression analysis and inhibition studies of a novel gene encoding Bowman-Birk type protease inhibitor from rice bean

USDA-ARS?s Scientific Manuscript database

This paper presents the first study describing the isolation, cloning and characterization of a full length gene encoding Bowman-Birk protease inhibitor (RbTI) from rice bean (Vigna umbellata). A full-length protease inhibitor gene with complete open reading frame of 327bp encoding 109 amino acids w...

Expression analysis of kenaf cinnamate 4-hydroxylase (C4H) ortholog during developmental and stress responses

USDA-ARS?s Scientific Manuscript database

This study was conducted to clone and analyze the expression pattern of a C4H gene encoding cinnamate 4-hydroxylase from kenaf (Hibiscus cannabinus L.). A full-length C4H ortholog was cloned using degenerate primers and the RACE (rapid amplification of cDNA ends) method. The full-length C4H ortholog...

Expression of FSH receptor in the hamster ovary during perinatal development

PubMed Central

Chakraborty, Prabuddha; Roy, Shyamal K.

2014-01-01

FSH plays an important role in ovarian follicular development, and it functions via the G-protein coupled FSH receptor. The objectives of the present study were to determine if full-length FSHR mRNA and corresponding protein were expressed in fetal through postnatal hamster ovaries to explain the FSH-induced primordial follicle formation, and if FSH or estrogen (E) would affect the expression. A full-length and two alternately spliced FSHR transcripts were expressed from E14 through P20. The level of the full-length FSHR mRNA increased markedly through P7 before stabilizing at a lower level with the formation and activation of primordial follicles. A predicted 87kDa FSHR protein band was detected in fetal through P4 ovaries, but additional bands appeared as ovary developed. FSHR immunosignal was present in undifferentiated somatic cells and oocytes in early postnatal ovaries, but was granulosa cells specific after follicles formed. Both eCG and E significantly up-regulated full-length FSHR mRNA levels. Therefore, FSHR is expressed in the hamster ovary from the fetal life to account for FSH-induced primordial follicle formation and cAMP production. Further, FSH or E regulates the receptor expression. PMID:25462586

A new strategy for full-length Ebola virus glycoprotein expression in E.coli.

PubMed

Zai, Junjie; Yi, Yinhua; Xia, Han; Zhang, Bo; Yuan, Zhiming

2016-12-01

Ebola virus (EBOV) causes severe hemorrhagic fever in humans and non-human primates with high rates of fatality. Glycoprotein (GP) is the only envelope protein of EBOV, which may play a critical role in virus attachment and entry as well as stimulating host protective immune responses. However, the lack of expression of full-length GP in Escherichia coli hinders the further study of its function in viral pathogenesis. In this study, the vp40 gene was fused to the full-length gp gene and cloned into a prokaryotic expression vector. We showed that the VP40-GP and GP-VP40 fusion proteins could be expressed in E.coli at 16 °C. In addition, it was shown that the position of vp40 in the fusion proteins affected the yields of the fusion proteins, with a higher level of production of the fusion protein when vp40 was upstream of gp compared to when it was downstream. The results provide a strategy for the expression of a large quantity of EBOV full-length GP, which is of importance for further analyzing the relationship between the structure and function of GP and developing an antibody for the treatment of EBOV infection.

Shortening actin filaments cause force generation in actomyosin network to change from contractile to extensile

NASA Astrophysics Data System (ADS)

Kumar, Nitin; Gardel, Margaret

Motor proteins in conjunction with filamentous proteins convert biochemical energy into mechanical energy which serves a number of cellular processes including cell motility, force generation and intracellular cargo transport. In-vitro experiments suggest that the forces generated by kinesin motors on microtubule bundles are extensile in nature whereas myosin motors on actin filaments are contractile. It is not clear how qualitatively similar systems can show completely different behaviors in terms of the nature of force generation. In order to answer this question, we carry out in vitro experiments where we form quasi 2D filamentous actomyosin networks and vary the length of actin filaments by adding capping protein. We show that when filaments are much shorter than their typical persistence length (approximately 10 microns), the forces generated are extensile and we see active nematic defect propagation, as seen in the microtubule-kinesin system. Based on this observation, we claim that the rigidity of rods plays an important role in dictating the nature of force generation in such systems. In order to understand this transition, we selectively label individual filaments and find that longer filaments show considerable bending and buckling, making them difficult to slide and extend along their length.

Recombinational Cloning Using Gateway and In-Fusion Cloning Schemes

PubMed Central

Throop, Andrea L.; LaBaer, Joshua

2015-01-01

The comprehensive study of protein structure and function, or proteomics, depends on the obtainability of full-length cDNAs in species-specific expression vectors and subsequent functional analysis of the expressed protein. Recombinational cloning is a universal cloning technique based on site-specific recombination that is independent of the insert DNA sequence of interest, which differentiates this method from the classical restriction enzyme-based cloning methods. Recombinational cloning enables rapid and efficient parallel transfer of DNA inserts into multiple expression systems. This unit summarizes strategies for generating expression-ready clones using the most popular recombinational cloning technologies, including the commercially available Gateway® (Life Technologies) and In-Fusion® (Clontech) cloning technologies. PMID:25827088

LIFEdb: a database for functional genomics experiments integrating information from external sources, and serving as a sample tracking system

PubMed Central

Bannasch, Detlev; Mehrle, Alexander; Glatting, Karl-Heinz; Pepperkok, Rainer; Poustka, Annemarie; Wiemann, Stefan

2004-01-01

We have implemented LIFEdb (http://www.dkfz.de/LIFEdb) to link information regarding novel human full-length cDNAs generated and sequenced by the German cDNA Consortium with functional information on the encoded proteins produced in functional genomics and proteomics approaches. The database also serves as a sample-tracking system to manage the process from cDNA to experimental read-out and data interpretation. A web interface enables the scientific community to explore and visualize features of the annotated cDNAs and ORFs combined with experimental results, and thus helps to unravel new features of proteins with as yet unknown functions. PMID:14681468

Automated sample-preparation technologies in genome sequencing projects.

PubMed

Hilbert, H; Lauber, J; Lubenow, H; Düsterhöft, A

2000-01-01

A robotic workstation system (BioRobot 96OO, QIAGEN) and a 96-well UV spectrophotometer (Spectramax 250, Molecular Devices) were integrated in to the process of high-throughput automated sequencing of double-stranded plasmid DNA templates. An automated 96-well miniprep kit protocol (QIAprep Turbo, QIAGEN) provided high-quality plasmid DNA from shotgun clones. The DNA prepared by this procedure was used to generate more than two mega bases of final sequence data for two genomic projects (Arabidopsis thaliana and Schizosaccharomyces pombe), three thousand expressed sequence tags (ESTs) plus half a mega base of human full-length cDNA clones, and approximately 53,000 single reads for a whole genome shotgun project (Pseudomonas putida).

Single-Cell mRNA-Seq Using the Fluidigm C1 System and Integrated Fluidics Circuits.

PubMed

Gong, Haibiao; Do, Devin; Ramakrishnan, Ramesh

2018-01-01

Single-cell mRNA-seq is a valuable tool to dissect expression profiles and to understand the regulatory network of genes. Microfluidics is well suited for single-cell analysis owing both to the small volume of the reaction chambers and easiness of automation. Here we describe the workflow of single-cell mRNA-seq using C1 IFC, which can isolate and process up to 96 cells. Both on-chip procedure (lysis, reverse transcription, and preamplification PCR) and off-chip sequencing library preparation protocols are described. The workflow generates full-length mRNA information, which is more valuable compared to 3' end counting method for many applications.

The Lockheed alternate partial polarizer universal filter

NASA Technical Reports Server (NTRS)

Title, A. M.

1976-01-01

A tunable birefringent filter using an alternate partial polarizer design has been built. The filter has a transmission of 38% in polarized light. Its full width at half maximum is .09A at 5500A. It is tunable from 4500 to 8500A by means of stepping motor actuated rotating half wave plates and polarizers. Wave length commands and thermal compensation commands are generated by a PPD 11/10 minicomputer. The alternate partial polarizer universal filter is compared with the universal birefringent filter and the design techniques, construction methods, and filter performance are discussed in some detail. Based on the experience of this filter some conclusions regarding the future of birefringent filters are elaborated.

A Rapid Method for Engineering Recombinant Polioviruses or Other Enteroviruses.

PubMed

Bessaud, Maël; Pelletier, Isabelle; Blondel, Bruno; Delpeyroux, Francis

2016-01-01

The cloning of large enterovirus RNA sequences is labor-intensive because of the frequent instability in bacteria of plasmidic vectors containing the corresponding cDNAs. In order to circumvent this issue we have developed a PCR-based method that allows the generation of highly modified or chimeric full-length enterovirus genomes. This method relies on fusion PCR which enables the concatenation of several overlapping cDNA amplicons produced separately. A T7 promoter sequence added upstream the fusion PCR products allows its transcription into infectious genomic RNAs directly in transfected cells constitutively expressing the phage T7 RNA polymerase. This method permits the rapid recovery of modified viruses that can be subsequently amplified on adequate cell-lines.

Fishing and bottom water temperature as drivers of change in maximum shell length in Atlantic surfclams (Spisula solidissima)

NASA Astrophysics Data System (ADS)

Munroe, D. M.; Narváez, D. A.; Hennen, D.; Jacobson, L.; Mann, R.; Hofmann, E. E.; Powell, E. N.; Klinck, J. M.

2016-03-01

Maximum shell length of Atlantic surfclams (Spisula solidissima) on the Middle Atlantic Bight (MAB) continental shelf, obtained from federal fishery survey data from 1982-present, has decreased by 15-20 mm. Two potential causes of this decreasing trend, fishery removal of large animals and stress due to warming bottom temperatures, were investigated using an individual-based model for post-settlement surfclams and a fifty-year hindcast of bottom water temperatures on the MAB. Simulations showed that fishing and/or warming bottom water temperature can cause decreases in maximum surfclam shell length (body size) equivalent to those observed in the fished stock. Independently, either localized fishing rates of 20% or sustained bottom temperatures that are 2 °C warmer than average conditions generate the observed decrease in maximum shell length. However, these independent conditions represent extremes and are not sustained in the MAB. The combined effects of fishing and warmer temperatures can generate simulated length decreases that are similar to observed decreases. Interannual variability in bottom water temperatures can also generate fluctuations in simulated shell length of up to 20 mm over a period of 10-15 years. If the change in maximum size is not genotypic, simulations also suggest that shell size composition of surfclam populations can recover if conditions change; however, that recovery could take a decade to become evident.

Minimap2: pairwise alignment for nucleotide sequences.

PubMed

Li, Heng

2018-05-10

Recent advances in sequencing technologies promise ultra-long reads of ∼100 kilo bases (kb) in average, full-length mRNA or cDNA reads in high throughput and genomic contigs over 100 mega bases (Mb) in length. Existing alignment programs are unable or inefficient to process such data at scale, which presses for the development of new alignment algorithms. Minimap2 is a general-purpose alignment program to map DNA or long mRNA sequences against a large reference database. It works with accurate short reads of ≥ 100bp in length, ≥1kb genomic reads at error rate ∼15%, full-length noisy Direct RNA or cDNA reads, and assembly contigs or closely related full chromosomes of hundreds of megabases in length. Minimap2 does split-read alignment, employs concave gap cost for long insertions and deletions (INDELs) and introduces new heuristics to reduce spurious alignments. It is 3-4 times as fast as mainstream short-read mappers at comparable accuracy, and is ≥30 times faster than long-read genomic or cDNA mappers at higher accuracy, surpassing most aligners specialized in one type of alignment. https://github.com/lh3/minimap2. hengli@broadinstitute.org.

Biomechanical aspects of segmented arch mechanics combined with power arm for controlled anterior tooth movement: A three-dimensional finite element study.

PubMed

Ozaki, Hiroya; Tominaga, Jun-Ya; Hamanaka, Ryo; Sumi, Mayumi; Chiang, Pao-Chang; Tanaka, Motohiro; Koga, Yoshiyuki; Yoshida, Noriaki

2015-01-01

The porpose of this study was to determine the optimal length of power arms for achieving controlled anterior tooth movement in segmented arch mechanics combined with power arm. A three-dimensional finite element method was applied for the simulation of en masse anterior tooth retraction in segmented power arm mechanics. The type of tooth movement, namely, the location of center of rotation of the maxillary central incisor in association with power arm length, was calculated after the retraction force was applied. When a 0.017 × 0.022-in archwire was inserted into the 0.018-in slot bracket, bodily movement was obtained at 9.1 mm length of power arm, namely, at the level of 1.8 mm above the center of resistance. In case a 0.018 × 0.025-in full-size archwire was used, bodily movement of the tooth was produced at the power arm length of 7.0 mm, namely, at the level of 0.3 mm below the center of resistance. Segmented arch mechanics required shorter length of power arms for achieving any type of controlled anterior tooth movement as compared to sliding mechanics. Therefore, this space closing mechanics could be widely applied even for the patients whose gingivobuccal fold is shallow. The segmented arch mechanics combined with power arm could provide higher amount of moment-to-force ratio sufficient for controlled anterior tooth movement without generating friction, and vertical forces when applying retraction force parallel to the occlusal plane. It is, therefore, considered that the segmented power arm mechanics has a simple appliance design and allows more efficient and controllable tooth movement.

Giardia canis: ultrastructural analysis of G. canis trophozoites transfected with full length G. canis virus cDNA transcripts

USDA-ARS?s Scientific Manuscript database

Giardia canis virus (GCV) is a double-stranded RNA (dsRNA) virus of the family Totiviridae. In this study, the full-length cDNA of the G. canis virus was constructed in pPoly2/sfinot vector and RNA was transcribed in vitro. Virus-free G. canis trophozoites were transfected with in vitro transcribed ...

Correlation of the level of full-length CFTR transcript with pulmonary phenotype in patients carrying R117H and 1342-1,-2delAG mutations

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hamosh, A.; Cutting, G.R.; Oates, R.

The R117H mutation occurs on two chromosome backgrounds, one associated with a 7 thymidine tract (7T-R11H) in the splice-acceptor site of intron 8, the other with a 5 thymidine tract (5T-R117H). We examined exon 9 splicing efficiency in 5 patients of genotype R117H/{delta}F508 and one carrying 1342-1,-2delAG{delta}F508, an obligate exon 9 slice site mutation. Four patients carried R117H on a 7T background -- three adult men with congenital bilateral absence of the vas deferens and one adolescent female with pancreatitis and borderline sweat chloride concentration. The patient with R117H on a 5T background had pancreatic sufficient CF (PS-CF). The 1342-1,-2delAGmore » patient has classic pancreatic insufficient CF (PI-CF). cDNA was synthesized from total RNA extracted from nasal epithlial cells and analyzed for CFTR splicing by 35 cycle PCR using primers in exon 7 and 11. The quantity of full length transcript derived from the R117H or {delta}F508 alleles was assessed by allele-specific oligonucleotide hybridization. While 91.4% of transcript from the 5T-R117H allele was full-length, only 42.2% of CFTR transcript from the 5T-R117H allele was full length. Since CBAVD patients have no lung disease and PS-CF patients do, this indicates that the threshold of developing CF lung disease is crossed when the amount of CFTR transcript bearing R117H is reduced by half. Interestingly, 17.1% of transcript derived from the 1342-1,-2delAG allele (or 8.6% of total CFTR transcript) was normal and full length. This suggests that up to 9% of full length wild-type CFTR transcript may be inadequate to escape the lung disease of CF and that a 9 thymidine tract followed by AAC (the result of the AG deletion) can be used as a splice donor with 2-9% efficiency.« less

Construction and EST sequencing of full-length, drought stress cDNA libraries for common beans (Phaseolus vulgaris L.)

PubMed Central

2011-01-01

Background Common bean is an important legume crop with only a moderate number of short expressed sequence tags (ESTs) made with traditional methods. The goal of this research was to use full-length cDNA technology to develop ESTs that would overlap with the beginning of open reading frames and therefore be useful for gene annotation of genomic sequences. The library was also constructed to represent genes expressed under drought, low soil phosphorus and high soil aluminum toxicity. We also undertook comparisons of the full-length cDNA library to two previous non-full clone EST sets for common bean. Results Two full-length cDNA libraries were constructed: one for the drought tolerant Mesoamerican genotype BAT477 and the other one for the acid-soil tolerant Andean genotype G19833 which has been selected for genome sequencing. Plants were grown in three soil types using deep rooting cylinders subjected to drought and non-drought stress and tissues were collected from both roots and above ground parts. A total of 20,000 clones were selected robotically, half from each library. Then, nearly 10,000 clones from the G19833 library were sequenced with an average read length of 850 nucleotides. A total of 4,219 unigenes were identified consisting of 2,981 contigs and 1,238 singletons. These were functionally annotated with gene ontology terms and placed into KEGG pathways. Compared to other EST sequencing efforts in common bean, about half of the sequences were novel or represented the 5' ends of known genes. Conclusions The present full-length cDNA libraries add to the technological toolbox available for common bean and our sequencing of these clones substantially increases the number of unique EST sequences available for the common bean genome. All of this should be useful for both functional gene annotation, analysis of splice site variants and intron/exon boundary determination by comparison to soybean genes or with common bean whole-genome sequences. In addition the library has a large number of transcription factors and will be interesting for discovery and validation of drought or abiotic stress related genes in common bean. PMID:22118559

Mutation-specific effects on thin filament length in thin filament myopathy.

PubMed

Winter, Josine M de; Joureau, Barbara; Lee, Eun-Jeong; Kiss, Balázs; Yuen, Michaela; Gupta, Vandana A; Pappas, Christopher T; Gregorio, Carol C; Stienen, Ger J M; Edvardson, Simon; Wallgren-Pettersson, Carina; Lehtokari, Vilma-Lotta; Pelin, Katarina; Malfatti, Edoardo; Romero, Norma B; Engelen, Baziel G van; Voermans, Nicol C; Donkervoort, Sandra; Bönnemann, C G; Clarke, Nigel F; Beggs, Alan H; Granzier, Henk; Ottenheijm, Coen A C

2016-06-01

Thin filament myopathies are among the most common nondystrophic congenital muscular disorders, and are caused by mutations in genes encoding proteins that are associated with the skeletal muscle thin filament. Mechanisms underlying muscle weakness are poorly understood, but might involve the length of the thin filament, an important determinant of force generation. We investigated the sarcomere length-dependence of force, a functional assay that provides insights into the contractile strength of muscle fibers as well as the length of the thin filaments, in muscle fibers from 51 patients with thin filament myopathy caused by mutations in NEB, ACTA1, TPM2, TPM3, TNNT1, KBTBD13, KLHL40, and KLHL41. Lower force generation was observed in muscle fibers from patients of all genotypes. In a subset of patients who harbor mutations in NEB and ACTA1, the lower force was associated with downward shifted force-sarcomere length relations, indicative of shorter thin filaments. Confocal microscopy confirmed shorter thin filaments in muscle fibers of these patients. A conditional Neb knockout mouse model, which recapitulates thin filament myopathy, revealed a compensatory mechanism; the lower force generation that was associated with shorter thin filaments was compensated for by increasing the number of sarcomeres in series. This allowed muscle fibers to operate at a shorter sarcomere length and maintain optimal thin-thick filament overlap. These findings might provide a novel direction for the development of therapeutic strategies for thin filament myopathy patients with shortened thin filament lengths. Ann Neurol 2016;79:959-969. © 2016 American Neurological Association.

Performance of IUCN proxies for generation length.

PubMed

Fung, Han Chi; Waples, Robin S

2017-08-01

One of the criteria used by the International Union for Conservation of Nature (IUCN) to assess threat status is the rate of decline in abundance over 3 generations or 10 years, whichever is longer. The traditional method for calculating generation length (T) uses age-specific survival and fecundity, but these data are rarely available. Consequently, proxies that require less information are often used, which introduces potential biases. The IUCN recommends 2 proxies based on adult mortality rate, T̂d = α + 1/d, and reproductive life span, T̂z = α + z * RL, where α is age at first reproduction, d is adult mortality rate, RL is reproductive life span, and z is a coefficient derived from data for comparable species. We used published life tables for 78 animal and plant populations to evaluate precision and bias of these proxies by comparing T̂d and T̂z with true generation length. Mean error rates in estimating T were 31% for T̂d and 20% for T̂z, but error rates for T̂d were 16% when we subtracted 1 year ( T̂d( adj )=T̂d-1 ), as suggested by theory; T̂d( adj ) also provided largely unbiased estimates regardless of the true generation length. Performance of T̂z depends on compilation of detailed data for comparable species, but our results suggest taxonomy is not a reliable indicator of comparability. All 3 proxies depend heavily on a reliable estimate of age at first reproduction, as we illustrated with 2 test species. The relatively large mean errors for all proxies emphasized the importance of collecting the detailed life-history information necessary to calculate true generation length. Unfortunately, publication of such data is less common than it was decades ago. We identified generic patterns of age-specific change in vital rates that can be used to predict expected patterns of bias from applying T̂d( adj ). Published 2017. This article is a U.S. Government work and is in the public domain in the USA.

Manual for automatic generation of finite element models of spiral bevel gears in mesh

NASA Technical Reports Server (NTRS)

Bibel, G. D.; Reddy, S.; Kumar, A.

1994-01-01

The goal of this research is to develop computer programs that generate finite element models suitable for doing 3D contact analysis of faced milled spiral bevel gears in mesh. A pinion tooth and a gear tooth are created and put in mesh. There are two programs: Points.f and Pat.f to perform the analysis. Points.f is based on the equation of meshing for spiral bevel gears. It uses machine tool settings to solve for an N x M mesh of points on the four surfaces, pinion concave and convex, and gear concave and convex. Points.f creates the file POINTS.OUT, an ASCI file containing N x M points for each surface. (N is the number of node points along the length of the tooth, and M is nodes along the height.) Pat.f reads POINTS.OUT and creates the file tl.out. Tl.out is a series of PATRAN input commands. In addition to the mesh density on the tooth face, additional user specified variables are the number of finite elements through the thickness, and the number of finite elements along the tooth full fillet. A full fillet is assumed to exist for both the pinion and gear.

Reference standard of penile size and prevalence of buried penis in Japanese newborn male infants.

PubMed

Matsuo, Nobutake; Ishii, Tomohiro; Takayama, John I; Miwa, Masayuki; Hasegawa, Tomonobu

2014-01-01

The present study set forth the reference values for penile size and determined the prevalence of buried penis in Japanese full-term newborns. The stretched penile length was measured and the presence of buried penis was assessed at 1-7 days of age in 547 Japanese full-term newborn infants born between 2008 and 2012 in Tokyo. The stretched penile lengths were compared at 1-12 hours and 1-7 days of age in 63 infants and by two observers in 73 infants to estimate postnatal changes and interobserver variation, respectively. The mean stretched penile length was 3.06 cm (SD, 0.26; 95% confidence interval [CI], 3.04-3.08) and the mean ratio of penile length to body length was 6.24 × 100(-1) (SD, 0.55 × 100(-1)), both of which were significantly smaller than those in Caucasian newborn infants. Buried penis was identified in 20 of 547 infants (3.7%; 95% CI, 2.1-5.2%). The first measurements of penile length at 1-12 hours were significantly smaller than the next measurements at 1-7 days (95% CI of the difference, 0.22-0.34). The 95% CI for the limits of agreement in the penile lengths measured by the two observers was -0.58 to -0.40 for the lower limit and 0.33 to 0.51 for the upper limit. These findings indicate that the penile length should be assessed after 24 hours of age by the reference standard of the same ethnicity for identifying micropenis and that buried penis is not uncommon in Japanese full-term newborns.

Turbulence Generation in Combustion.

DTIC Science & Technology

1987-07-22

flame length . This work is summarized in this section. I1.1 Model for Turbulent Burning Velocity For a range of turbulence conditions including...Variable density effects have been added in an approximation, and an expression for the length of jet flames has been developed. The flame length expression...of jet mixing and jet flame length data using fractals, College of Engineering, Energy Report E-86-02, Comell University, Ithaca, NY, 1986. Results

Construction and Evaluation of Normalized cDNA Libraries Enriched with Full-Length Sequences for Rapid Discovery of New Genes from Sisal (Agave sisalana Perr.) Different Developmental Stages

PubMed Central

Zhou, Wen-Zhao; Zhang, Yan-Mei; Lu, Jun-Ying; Li, Jun-Feng

2012-01-01

To provide a resource of sisal-specific expressed sequence data and facilitate this powerful approach in new gene research, the preparation of normalized cDNA libraries enriched with full-length sequences is necessary. Four libraries were produced with RNA pooled from Agave sisalana multiple tissues to increase efficiency of normalization and maximize the number of independent genes by SMART™ method and the duplex-specific nuclease (DSN). This procedure kept the proportion of full-length cDNAs in the subtracted/normalized libraries and dramatically enhanced the discovery of new genes. Sequencing of 3875 cDNA clones of libraries revealed 3320 unigenes with an average insert length about 1.2 kb, indicating that the non-redundancy of libraries was about 85.7%. These unigene functions were predicted by comparing their sequences to functional domain databases and extensively annotated with Gene Ontology (GO) terms. Comparative analysis of sisal unigenes and other plant genomes revealed that four putative MADS-box genes and knotted-like homeobox (knox) gene were obtained from a total of 1162 full-length transcripts. Furthermore, real-time PCR showed that the characteristics of their transcripts mainly depended on the tight expression regulation of a number of genes during the leaf and flower development. Analysis of individual library sequence data indicated that the pooled-tissue approach was highly effective in discovering new genes and preparing libraries for efficient deep sequencing. PMID:23202944

Wave generation by fracture initiation and propagation in geomaterials with internal rotations

NASA Astrophysics Data System (ADS)

Esin, Maxim; Pasternak, Elena; Dyskin, Arcady; Xu, Yuan

2016-04-01

Crack or fracture initiation and propagation in geomaterials are sources of waves and is important in both stability and fracture (e.g. hydraulic fracture) monitoring. Many geomaterials consist of particles or other constituents capable of rotating with respect to each other, either due to the absence of the binder phase (fragmented materials) or due to extensive damage of the cement between the constituents inflicted by previous loading. In investigating the wave generated in fracturing it is important to distinguish between the cases when the fracture is instantaneously initiated to its full length or propagates from a smaller initial crack. We show by direct physical experiments and discrete element modelling of 2D arrangements of unbonded disks that under compressive load fractures are initiated instantaneously as a result of the material instability and localisation. Such fractures generate waves as a single impulse impact. When the fractures propagate, they produce a sequence of impulses associated with the propagation steps. This manifests itself as acoustic (microseismic) emission whose temporal pattern contains the information of the fracture geometry, such as fractal dimension of the fracture. The description of this process requires formulating criteria of crack growth capable of taking into account the internal rotations. We developed an analytical solution based on the Cosserat continuum where each point of body has three translational and three rotational degrees of freedom. When the Cosserat characteristic lengths are comparable with the grain sizes, the simplified equations of small-scale Cosserat continuum can be used. We established that the order of singularity of the main asymptotic term for moment stress is higher than the order of singularity for conventional stress. Therefore, the mutual rotation of particles and related bending and/or twisting of the bonds between the particles represent an unconventional mechanism of crack propagation.

Improved maize reference genome with single-molecule technologies.

PubMed

Jiao, Yinping; Peluso, Paul; Shi, Jinghua; Liang, Tiffany; Stitzer, Michelle C; Wang, Bo; Campbell, Michael S; Stein, Joshua C; Wei, Xuehong; Chin, Chen-Shan; Guill, Katherine; Regulski, Michael; Kumari, Sunita; Olson, Andrew; Gent, Jonathan; Schneider, Kevin L; Wolfgruber, Thomas K; May, Michael R; Springer, Nathan M; Antoniou, Eric; McCombie, W Richard; Presting, Gernot G; McMullen, Michael; Ross-Ibarra, Jeffrey; Dawe, R Kelly; Hastie, Alex; Rank, David R; Ware, Doreen

2017-06-22

Complete and accurate reference genomes and annotations provide fundamental tools for characterization of genetic and functional variation. These resources facilitate the determination of biological processes and support translation of research findings into improved and sustainable agricultural technologies. Many reference genomes for crop plants have been generated over the past decade, but these genomes are often fragmented and missing complex repeat regions. Here we report the assembly and annotation of a reference genome of maize, a genetic and agricultural model species, using single-molecule real-time sequencing and high-resolution optical mapping. Relative to the previous reference genome, our assembly features a 52-fold increase in contig length and notable improvements in the assembly of intergenic spaces and centromeres. Characterization of the repetitive portion of the genome revealed more than 130,000 intact transposable elements, allowing us to identify transposable element lineage expansions that are unique to maize. Gene annotations were updated using 111,000 full-length transcripts obtained by single-molecule real-time sequencing. In addition, comparative optical mapping of two other inbred maize lines revealed a prevalence of deletions in regions of low gene density and maize lineage-specific genes.

C-Terminal End-Directed Protein Elimination by CRL2 Ubiquitin Ligases.

PubMed

Lin, Hsiu-Chuan; Yeh, Chi-Wei; Chen, Yen-Fu; Lee, Ting-Ting; Hsieh, Pei-Yun; Rusnac, Domnita V; Lin, Sung-Ya; Elledge, Stephen J; Zheng, Ning; Yen, Hsueh-Chi S

2018-05-17

The proteolysis-assisted protein quality control system guards the proteome from potentially detrimental aberrant proteins. How miscellaneous defective proteins are specifically eliminated and which molecular characteristics direct them for removal are fundamental questions. We reveal a mechanism, DesCEND (destruction via C-end degrons), by which CRL2 ubiquitin ligase uses interchangeable substrate receptors to recognize the unusual C termini of abnormal proteins (i.e., C-end degrons). C-end degrons are mostly less than ten residues in length and comprise a few indispensable residues along with some rather degenerate ones. The C-terminal end position is essential for C-end degron function. Truncated selenoproteins generated by translation errors and the USP1 N-terminal fragment from post-translational cleavage are eliminated by DesCEND. DesCEND also targets full-length proteins with naturally occurring C-end degrons. The C-end degron in DesCEND echoes the N-end degron in the N-end rule pathway, highlighting the dominance of protein "ends" as indicators for protein elimination. Copyright © 2018 Elsevier Inc. All rights reserved.

An experimental investigation of thermoacoustic lasers operating in audible frequency range

NASA Astrophysics Data System (ADS)

Kolhe, Sanket Anil

Thermoacoustic lasers convert heat from a high-temperature heat source into acoustic power while rejecting waste heat to a low temperature sink. The working fluids involved can be air or noble gases which are nontoxic and environmentally benign. Simple in construction due to absence of moving parts, thermoacoustic lasers can be employed to achieve generation of electricity at individual homes, water-heating for domestic purposes, and to facilitate space heating and cooling. The possibility of utilizing waste heat or solar energy to run thermoacoustic devices makes them technically promising and economically viable to generate large quantities of acoustic energy. The research presented in this thesis deals with the effects of geometric parameters (stack position, stack length, tube length) associated with a thermoacoustic laser on the output sound wave. The effects of varying input power on acoustic output were also studied. Based on the experiments, optimum operating conditions were identified and qualitative and/or quantitative explanations were provided to justify our observations. It was observed that the maximum sound pressure level was generated for the laser with the stack positioned at a distance of quarter lengths of a resonator from the closed end. Higher sound pressure levels were recorded for the laser with longer stack lengths and longer resonator lengths. Efforts were also made to develop high-frequency thermoacoustic lasers.

Massive Collection of Full-Length Complementary DNA Clones and Microarray Analyses:. Keys to Rice Transcriptome Analysis

NASA Astrophysics Data System (ADS)

Kikuchi, Shoshi

2009-02-01

Completion of the high-precision genome sequence analysis of rice led to the collection of about 35,000 full-length cDNA clones and the determination of their complete sequences. Mapping of these full-length cDNA sequences has given us information on (1) the number of genes expressed in the rice genome; (2) the start and end positions and exon-intron structures of rice genes; (3) alternative transcripts; (4) possible encoded proteins; (5) non-protein-coding (np) RNAs; (6) the density of gene localization on the chromosome; (7) setting the parameters of gene prediction programs; and (8) the construction of a microarray system that monitors global gene expression. Manual curation for rice gene annotation by using mapping information on full-length cDNA and EST assemblies has revealed about 32,000 expressed genes in the rice genome. Analysis of major gene families, such as those encoding membrane transport proteins (pumps, ion channels, and secondary transporters), along with the evolution from bacteria to higher animals and plants, reveals how gene numbers have increased through adaptation to circumstances. Family-based gene annotation also gives us a new way of comparing organisms. Massive amounts of data on gene expression under many kinds of physiological conditions are being accumulated in rice oligoarrays (22K and 44K) based on full-length cDNA sequences. Cluster analyses of genes that have the same promoter cis-elements, that have similar expression profiles, or that encode enzymes in the same metabolic pathways or signal transduction cascades give us clues to understanding the networks of gene expression in rice. As a tool for that purpose, we recently developed "RiCES", a tool for searching for cis-elements in the promoter regions of clustered genes.

Cost-Effective Sequencing of Full-Length cDNA Clones Powered by a De Novo-Reference Hybrid Assembly

PubMed Central

Sugano, Sumio; Morishita, Shinichi; Suzuki, Yutaka

2010-01-01

Background Sequencing full-length cDNA clones is important to determine gene structures including alternative splice forms, and provides valuable resources for experimental analyses to reveal the biological functions of coded proteins. However, previous approaches for sequencing cDNA clones were expensive or time-consuming, and therefore, a fast and efficient sequencing approach was demanded. Methodology We developed a program, MuSICA 2, that assembles millions of short (36-nucleotide) reads collected from a single flow cell lane of Illumina Genome Analyzer to shotgun-sequence ∼800 human full-length cDNA clones. MuSICA 2 performs a hybrid assembly in which an external de novo assembler is run first and the result is then improved by reference alignment of shotgun reads. We compared the MuSICA 2 assembly with 200 pooled full-length cDNA clones finished independently by the conventional primer-walking using Sanger sequencers. The exon-intron structure of the coding sequence was correct for more than 95% of the clones with coding sequence annotation when we excluded cDNA clones insufficiently represented in the shotgun library due to PCR failure (42 out of 200 clones excluded), and the nucleotide-level accuracy of coding sequences of those correct clones was over 99.99%. We also applied MuSICA 2 to full-length cDNA clones from Toxoplasma gondii, to confirm that its ability was competent even for non-human species. Conclusions The entire sequencing and shotgun assembly takes less than 1 week and the consumables cost only ∼US$3 per clone, demonstrating a significant advantage over previous approaches. PMID:20479877

A Possible Role of the Full-Length Nascent Protein in Post-Translational Ribosome Recycling.

PubMed

Das, Debasis; Samanta, Dibyendu; Bhattacharya, Arpita; Basu, Arunima; Das, Anindita; Ghosh, Jaydip; Chakrabarti, Abhijit; Das Gupta, Chanchal

2017-01-01

Each cycle of translation initiation in bacterial cell requires free 50S and 30S ribosomal subunits originating from the post-translational dissociation of 70S ribosome from the previous cycle. Literature shows stable dissociation of 70S from model post-termination complexes by the concerted action of Ribosome Recycling Factor (RRF) and Elongation Factor G (EF-G) that interact with the rRNA bridge B2a/B2b joining 50S to 30S. In such experimental models, the role of full-length nascent protein was never considered seriously. We observed relatively slow release of full-length nascent protein from 50Sof post translation ribosome, and in that process, its toe prints on the rRNA in vivo and in in vitro translation with E.coli S30 extract. We reported earlier that a number of chemically unfolded proteins like bovine carbonic anhydrase (BCA), lactate dehydrogenase (LDH), malate dehydrogenase (MDH), lysozyme, ovalbumin etc., when added to free 70Sin lieu of the full length nascent proteins, also interact with identical RNA regions of the 23S rRNA. Interestingly the rRNA nucleotides that slow down release of the C-terminus of full-length unfolded protein were found in close proximity to the B2a/B2b bridge. It indicated a potentially important chemical reaction conserved throughout the evolution. Here we set out to probe that conserved role of unfolded protein conformation in splitting the free or post-termination 70S. How both the RRF-EFG dependent and the plausible nascent protein-EFG dependent ribosome recycling pathways might be relevant in bacteria is discussed here.

A Possible Role of the Full-Length Nascent Protein in Post-Translational Ribosome Recycling

PubMed Central

Das, Debasis; Samanta, Dibyendu; Bhattacharya, Arpita; Basu, Arunima; Das, Anindita; Ghosh, Jaydip; Chakrabarti, Abhijit; Das Gupta, Chanchal

2017-01-01

Each cycle of translation initiation in bacterial cell requires free 50S and 30S ribosomal subunits originating from the post-translational dissociation of 70S ribosome from the previous cycle. Literature shows stable dissociation of 70S from model post-termination complexes by the concerted action of Ribosome Recycling Factor (RRF) and Elongation Factor G (EF-G) that interact with the rRNA bridge B2a/B2b joining 50S to 30S. In such experimental models, the role of full-length nascent protein was never considered seriously. We observed relatively slow release of full-length nascent protein from 50Sof post translation ribosome, and in that process, its toe prints on the rRNA in vivo and in in vitro translation with E.coli S30 extract. We reported earlier that a number of chemically unfolded proteins like bovine carbonic anhydrase (BCA), lactate dehydrogenase (LDH), malate dehydrogenase (MDH), lysozyme, ovalbumin etc., when added to free 70Sin lieu of the full length nascent proteins, also interact with identical RNA regions of the 23S rRNA. Interestingly the rRNA nucleotides that slow down release of the C-terminus of full-length unfolded protein were found in close proximity to the B2a/B2b bridge. It indicated a potentially important chemical reaction conserved throughout the evolution. Here we set out to probe that conserved role of unfolded protein conformation in splitting the free or post-termination 70S. How both the RRF-EFG dependent and the plausible nascent protein–EFG dependent ribosome recycling pathways might be relevant in bacteria is discussed here. PMID:28099529

Native Mutant Huntingtin in Human Brain

PubMed Central

Sapp, Ellen; Valencia, Antonio; Li, Xueyi; Aronin, Neil; Kegel, Kimberly B.; Vonsattel, Jean-Paul; Young, Anne B.; Wexler, Nancy; DiFiglia, Marian

2012-01-01

Huntington disease (HD) is caused by polyglutamine expansion in the N terminus of huntingtin (htt). Analysis of human postmortem brain lysates by SDS-PAGE and Western blot reveals htt as full-length and fragmented. Here we used Blue Native PAGE (BNP) and Western blots to study native htt in human postmortem brain. Antisera against htt detected a single band broadly migrating at 575–850 kDa in control brain and at 650–885 kDa in heterozygous and Venezuelan homozygous HD brains. Anti-polyglutamine antisera detected full-length mutant htt in HD brain. There was little htt cleavage even if lysates were pretreated with trypsin, indicating a property of native htt to resist protease cleavage. A soluble mutant htt fragment of about 180 kDa was detected with anti-htt antibody Ab1 (htt-(1–17)) and increased when lysates were treated with denaturants (SDS, 8 m urea, DTT, or trypsin) before BNP. Wild-type htt was more resistant to denaturants. Based on migration of in vitro translated htt fragments, the 180-kDa segment terminated ≈htt 670–880 amino acids. If second dimension SDS-PAGE followed BNP, the 180-kDa mutant htt was absent, and 43–50 kDa htt fragments appeared. Brain lysates from two HD mouse models expressed native full-length htt; a mutant fragment formed if lysates were pretreated with 8 m urea + DTT. Native full-length mutant htt in embryonic HD140Q/140Q mouse primary neurons was intact during cell death and when cell lysates were exposed to denaturants before BNP. Thus, native mutant htt occurs in brain and primary neurons as a soluble full-length monomer. PMID:22375012

Structural modulation of factor VIIa by full-length tissue factor (TF1-263): implication of novel interactions between EGF2 domain and TF.

PubMed

Prasad, Ramesh; Sen, Prosenjit

2018-02-01

Tissue factor (TF)-mediated factor VII (FVII) activation and a subsequent proteolytic TF-FVIIa binary complex formation is the key step initiating the coagulation cascade, with implications in various homeostatic and pathologic scenarios. TF binding allosterically modifies zymogen-like free FVIIa to its highly catalytically active form. As a result of unresolved crystal structure of the full-length TF 1-263 -FVIIa binary complex and free FVIIa, allosteric alterations in FVIIa following its binding to full-length TF and the consequences of these on function are not entirely clear. The present study aims to map and identify structural alterations in FVIIa and TF resulting from full-length TF binding to FVIIa and the key events responsible for enhanced FVIIa activity in coagulation. We constructed the full-length TF 1-263 -FVIIa membrane bound complex using computational modeling and subjected it to molecular dynamics (MD) simulations. MD simulations showed that TF alters the structure of each domain of FVIIa and these combined alterations contribute to enhanced TF-FVIIa activity. Detailed, domain-wise investigation revealed several new non-covalent interactions between TF and FVIIa that were not found in the truncated soluble TF-FVIIa crystal structure. The structural modulation of each FVIIa domain imparted by TF indicated that both inter and intra-domain communication is crucial for allosteric modulation of FVIIa. Our results suggest that these newly formed interactions can provide additional stability to the protease domain and regulate its activity profile by governing catalytic triad (CT) orientation and localization. The unexplored newly formed interactions between EGF2 and TF provides a possible explanation for TF-induced allosteric activation of FVIIa.

Molecular Cloning and Characterization of Two Pig Vasoactive Intestinal Polypeptide Receptors (VPAC1-R and VPAC2-R)

PubMed Central

He, Xiaping; Meng, Fengyan; Wang, Yajun

2014-01-01

We here report the cloning, tissue expression, and functional analyses of the two pig vasoactive intestinal polypeptide (VIP) receptors (pVPAC1-R and pVPAC2-R). The cloned full-length pVPAC1-R and pVPAC2-R share high structural similarity with their mammalian counterparts. Functional assay revealed that the full-length pVPAC1-R and pVPAC2-R-expressed Chinese hamster ovary (CHO) cells could be activated by pVIP and pPACAP38 potently, indicating that pVPAC1-R and pVPAC2-R are capable of binding VIP and pituitary adenylate cyclase-activating polypeptide (PACAP). In addition to the identification of the transcripts encoding the two full-length receptors, multiple splice transcript variants were isolated. Comparison with the pig genome database revealed that pVPAC1-R and pVPAC2-R share a unique gene structure with 14 exons different from other vertebrates. Reverse transcription and polymerase chain reaction (RT-PCR) assays further showed that the transcript encoding the full-length pVPAC2-R is widely expressed in all adult tissues whereas the splice variants of pVPAC1-R are predominantly expressed in all tissues instead of the transcript encoding the full-length receptor, hinting that pVPAC2-R may play more important roles than pVPAC1-R in mediating VIP and PACAP actions. Our present findings help to elucidate the important role of VIP and PACAP and promote to rethink of their species-specific physiological roles including their actions in regulation of phenotypic traits in pigs. PMID:24520933

Novel full-length major histocompatibility complex class I allele discovery and haplotype definition in pig-tailed macaques.

PubMed

Semler, Matthew R; Wiseman, Roger W; Karl, Julie A; Graham, Michael E; Gieger, Samantha M; O'Connor, David H

2018-06-01

Pig-tailed macaques (Macaca nemestrina, Mane) are important models for human immunodeficiency virus (HIV) studies. Their infectability with minimally modified HIV makes them a uniquely valuable animal model to mimic human infection with HIV and progression to acquired immunodeficiency syndrome (AIDS). However, variation in the pig-tailed macaque major histocompatibility complex (MHC) and the impact of individual transcripts on the pathogenesis of HIV and other infectious diseases is understudied compared to that of rhesus and cynomolgus macaques. In this study, we used Pacific Biosciences single-molecule real-time circular consensus sequencing to describe full-length MHC class I (MHC-I) transcripts for 194 pig-tailed macaques from three breeding centers. We then used the full-length sequences to infer Mane-A and Mane-B haplotypes containing groups of MHC-I transcripts that co-segregate due to physical linkage. In total, we characterized full-length open reading frames (ORFs) for 313 Mane-A, Mane-B, and Mane-I sequences that defined 86 Mane-A and 106 Mane-B MHC-I haplotypes. Pacific Biosciences technology allows us to resolve these Mane-A and Mane-B haplotypes to the level of synonymous allelic variants. The newly defined haplotypes and transcript sequences containing full-length ORFs provide an important resource for infectious disease researchers as certain MHC haplotypes have been shown to provide exceptional control of simian immunodeficiency virus (SIV) replication and prevention of AIDS-like disease in nonhuman primates. The increased allelic resolution provided by Pacific Biosciences sequencing also benefits transplant research by allowing researchers to more specifically match haplotypes between donors and recipients to the level of nonsynonymous allelic variation, thus reducing the risk of graft-versus-host disease.

OmpA: A Flexible Clamp for Bacterial Cell Wall Attachment.

PubMed

Samsudin, Firdaus; Ortiz-Suarez, Maite L; Piggot, Thomas J; Bond, Peter J; Khalid, Syma

2016-12-06

The envelope of Gram-negative bacteria is highly complex, containing separate outer and inner membranes and an intervening periplasmic space encompassing a peptidoglycan (PGN) cell wall. The PGN scaffold is anchored non-covalently to the outer membrane via globular OmpA-like domains of various proteins. We report atomically detailed simulations of PGN bound to OmpA in three different states, including the isolated C-terminal domain (CTD), the full-length monomer, or the complete full-length dimeric form. Comparative analysis of dynamics of OmpA CTD from different bacteria helped to identify a conserved PGN-binding mode. The dynamics of full-length OmpA, embedded within a realistic representation of the outer membrane containing full-rough (Ra) lipopolysaccharide, phospholipids, and cardiolipin, suggested how the protein may provide flexible mechanical support to the cell wall. An accurate model of the heterogeneous bacterial cell envelope should facilitate future efforts to develop antibacterial agents. Copyright © 2016 Elsevier Ltd. All rights reserved.

Two distinct extracellular RNA signatures released by a single cell type identified by microarray and next-generation sequencing

PubMed Central

Lässer, Cecilia; Shelke, Ganesh Vilas; Yeri, Ashish; Kim, Dae-Kyum; Crescitelli, Rossella; Raimondo, Stefania; Sjöstrand, Margareta; Gho, Yong Song; Van Keuren Jensen, Kendall; Lötvall, Jan

2017-01-01

ABSTRACT Cells secrete extracellular RNA (exRNA) to their surrounding environment and exRNA has been found in many body fluids such as blood, breast milk and cerebrospinal fluid. However, there are conflicting results regarding the nature of exRNA. Here, we have separated 2 distinct exRNA profiles released by mast cells, here termed high-density (HD) and low-density (LD) exRNA. The exRNA in both fractions was characterized by microarray and next-generation sequencing. Both exRNA fractions contained mRNA and miRNA, and the mRNAs in the LD exRNA correlated closely with the cellular mRNA, whereas the HD mRNA did not. Furthermore, the HD exRNA was enriched in lincRNA, antisense RNA, vault RNA, snoRNA, and snRNA with little or no evidence of full-length 18S and 28S rRNA. The LD exRNA was enriched in mitochondrial rRNA, mitochondrial tRNA, tRNA, piRNA, Y RNA, and full-length 18S and 28S rRNA. The proteomes of the HD and LD exRNA-containing fractions were determined with LC-MS/MS and analyzed with Gene Ontology term finder, which showed that both proteomes were associated with the term extracellular vesicles and electron microscopy suggests that at least a part of the exRNA is associated with exosome-like extracellular vesicles. Additionally, the proteins in the HD fractions tended to be associated with the nucleus and ribosomes, whereas the LD fraction proteome tended to be associated with the mitochondrion. We show that the 2 exRNA signatures released by a single cell type can be separated by floatation on a density gradient. These results show that cells can release multiple types of exRNA with substantial differences in RNA species content. This is important for any future studies determining the nature and function of exRNA released from different cells under different conditions. PMID:27791479

Frame synchronization performance and analysis

NASA Technical Reports Server (NTRS)

Aguilera, C. S. R.; Swanson, L.; Pitt, G. H., III

1988-01-01

The analysis used to generate the theoretical models showing the performance of the frame synchronizer is described for various frame lengths and marker lengths at various signal to noise ratios and bit error tolerances.

Measurement of the full-field polymerization shrinkage and depth of cure of dental composites using digital image correlation.

PubMed

Li, Jianying; Fok, Alex S L; Satterthwaite, Julian; Watts, David C

2009-05-01

The aim of this study was to measure the full-field polymerization shrinkage of dental composites using optical image correlation method. Bar specimens of cross-section 4mm x 2mm and length 10mm approximately were light cured with two irradiances, 450 mW/cm(2) and 180 mW/cm(2), respectively. The curing light was generated with Optilux 501 (Kerr) and the two different irradiances were achieved by adjusting the distance between the light tip and the specimen. A single-camera 2D measuring system was used to record the deformation of the composite specimen for 30 min at a frequency of 0.1 Hz. The specimen surface under observation was sprayed with paint to produce sufficient contrast to allow tracking of individual points on the surface. The curing light was applied to one end of the specimen for 40s during which the painted surface was fully covered. After curing, the cover was removed immediately so that deformation of the painted surface could be recorded by the camera. The images were then analyzed with specialist software and the volumetric shrinkage determined along the beam length. A typical shrinkage strain field obtained on a specimen surface was highly non-uniform, even at positions of constant distance from the irradiation surface, indicating possible heterogeneity in material composition and shrinkage behavior in the composite. The maximum volumetric shrinkage strain of approximately 1.5% occurred at a subsurface distance of about 1mm, instead of at the irradiation surface. After reaching its peak value, the shrinkage strain then gradually decreased with increasing distance along the beam length, before leveling off to a value of approximately 0.2% at a distance of 4-5mm. The maximum volumetric shrinkage obtained agreed well with the value of 1.6% reported by the manufacturer for the composite examined in this work. Using irradiance of 180 mW/cm(2) resulted in only slightly less polymerization shrinkage than using irradiance of 450 mW/cm(2). Compared to the other measurement methods, the image correlation method is capable of producing full-field information about the polymerization shrinkage behavior of dental composites.

Optimization of the Heat Exchangers of a Thermoelectric Generation System

NASA Astrophysics Data System (ADS)

Martínez, A.; Vián, J. G.; Astrain, D.; Rodríguez, A.; Berrio, I.

2010-09-01

The thermal resistances of the heat exchangers have a strong influence on the electric power produced by a thermoelectric generator. In this work, the heat exchangers of a thermoelectric generator have been optimized in order to maximize the electric power generated. This thermoelectric generator harnesses heat from the exhaust gas of a domestic gas boiler. Statistical design of experiments was used to assess the influence of five factors on both the electric power generated and the pressure drop in the chimney: height of the generator, number of modules per meter of generator height, length of the fins of the hot-side heat exchanger (HSHE), length of the gap between fins of the HSHE, and base thickness of the HSHE. The electric power has been calculated using a computational model, whereas Fluent computational fluid dynamics (CFD) has been used to obtain the thermal resistances of the heat exchangers and the pressure drop. Finally, the thermoelectric generator has been optimized, taking into account the restrictions on the pressure drop.

Efficiently measuring dimensions of the externalizing spectrum model: Development of the Externalizing Spectrum Inventory-Computerized Adaptive Test (ESI-CAT).

PubMed

Sunderland, Matthew; Slade, Tim; Krueger, Robert F; Markon, Kristian E; Patrick, Christopher J; Kramer, Mark D

2017-07-01

The development of the Externalizing Spectrum Inventory (ESI) was motivated by the need to comprehensively assess the interrelated nature of externalizing psychopathology and personality using an empirically driven framework. The ESI measures 23 theoretically distinct yet related unidimensional facets of externalizing, which are structured under 3 superordinate factors representing general externalizing, callous aggression, and substance abuse. One limitation of the ESI is its length at 415 items. To facilitate the use of the ESI in busy clinical and research settings, the current study sought to examine the efficiency and accuracy of a computerized adaptive version of the ESI. Data were collected over 3 waves and totaled 1,787 participants recruited from undergraduate psychology courses as well as male and female state prisons. A series of 6 algorithms with different termination rules were simulated to determine the efficiency and accuracy of each test under 3 different assumed distributions. Scores generated using an optimal adaptive algorithm evidenced high correlations (r > .9) with scores generated using the full ESI, brief ESI item-based factor scales, and the 23 facet scales. The adaptive algorithms for each facet administered a combined average of 115 items, a 72% decrease in comparison to the full ESI. Similarly, scores on the item-based factor scales of the ESI-brief form (57 items) were generated using on average of 17 items, a 70% decrease. The current study successfully demonstrates that an adaptive algorithm can generate similar scores for the ESI and the 3 item-based factor scales using a fraction of the total item pool. (PsycINFO Database Record (c) 2017 APA, all rights reserved).

Identification of gyrB and rpoB gene mutations and differentially expressed proteins between a novobiocin-resistant Aeromonas hydrophila catfish vaccine strain and its virulent parent strain

USDA-ARS?s Scientific Manuscript database

Sequence comparison between the full-length 2412 bp DNA gyrase subunit B (gyrB) gene of a novobiocin resistant Aeromonas hydrophila AH11NOVO vaccine strain and that of its virulent parent strain AH11P revealed 10 missense mutations. Similarly, sequence comparison between the full-length 4092 bp RNA ...

A Novel Domain Assembly Routine for Creating Full-Length Models of Membrane Proteins from Known Domain Structures.

PubMed

Koehler Leman, Julia; Bonneau, Richard

2018-04-03

Membrane proteins composed of soluble and membrane domains are often studied one domain at a time. However, to understand the biological function of entire protein systems and their interactions with each other and drugs, knowledge of full-length structures or models is required. Although few computational methods exist that could potentially be used to model full-length constructs of membrane proteins, none of these methods are perfectly suited for the problem at hand. Existing methods require an interface or knowledge of the relative orientations of the domains or are not designed for domain assembly, and none of them are developed for membrane proteins. Here we describe the first domain assembly protocol specifically designed for membrane proteins that assembles intra- and extracellular soluble domains and the transmembrane domain into models of the full-length membrane protein. Our protocol does not require an interface between the domains and samples possible domain orientations based on backbone dihedrals in the flexible linker regions, created via fragment insertion, while keeping the transmembrane domain fixed in the membrane. For five examples tested, our method mp_domain_assembly, implemented in RosettaMP, samples domain orientations close to the known structure and is best used in conjunction with experimental data to reduce the conformational search space.

Large-scale identification and characterization of alternative splicing variants of human gene transcripts using 56 419 completely sequenced and manually annotated full-length cDNAs

PubMed Central

Takeda, Jun-ichi; Suzuki, Yutaka; Nakao, Mitsuteru; Barrero, Roberto A.; Koyanagi, Kanako O.; Jin, Lihua; Motono, Chie; Hata, Hiroko; Isogai, Takao; Nagai, Keiichi; Otsuki, Tetsuji; Kuryshev, Vladimir; Shionyu, Masafumi; Yura, Kei; Go, Mitiko; Thierry-Mieg, Jean; Thierry-Mieg, Danielle; Wiemann, Stefan; Nomura, Nobuo; Sugano, Sumio; Gojobori, Takashi; Imanishi, Tadashi

2006-01-01

We report the first genome-wide identification and characterization of alternative splicing in human gene transcripts based on analysis of the full-length cDNAs. Applying both manual and computational analyses for 56 419 completely sequenced and precisely annotated full-length cDNAs selected for the H-Invitational human transcriptome annotation meetings, we identified 6877 alternative splicing genes with 18 297 different alternative splicing variants. A total of 37 670 exons were involved in these alternative splicing events. The encoded protein sequences were affected in 6005 of the 6877 genes. Notably, alternative splicing affected protein motifs in 3015 genes, subcellular localizations in 2982 genes and transmembrane domains in 1348 genes. We also identified interesting patterns of alternative splicing, in which two distinct genes seemed to be bridged, nested or having overlapping protein coding sequences (CDSs) of different reading frames (multiple CDS). In these cases, completely unrelated proteins are encoded by a single locus. Genome-wide annotations of alternative splicing, relying on full-length cDNAs, should lay firm groundwork for exploring in detail the diversification of protein function, which is mediated by the fast expanding universe of alternative splicing variants. PMID:16914452

Full-length genome sequences of five hepatitis C virus isolates representing subtypes 3g, 3h, 3i and 3k, and a unique genotype 3 variant.

PubMed

Lu, Ling; Li, Chunhua; Yuan, Jie; Lu, Teng; Okamoto, Hiroaki; Murphy, Donald G

2013-03-01

We characterized the full-length genomes of five distinct hepatitis C virus (HCV)-3 isolates. These represent the first complete genomes for subtypes 3g and 3h, the second such genomes for 3k and 3i, and of one novel variant presently not assigned to a subtype. Each genome was determined from 18-25 overlapping fragments. They had lengths of 9579-9660 nt and each contained a single ORF encoding 3020-3025 aa. They were isolated from five patients residing in Canada; four were of Asian origin and one was of Somali origin. Phylogenetic analysis using 64 partial NS5B sequences differentiated 10 assigned subtypes, 3a-3i and 3k, and two additional lineages within genotype 3. From the data of this study, HCV-3 full-length sequences are now available for six of the assigned subtypes and one unassigned. Our findings should add insights to HCV evolutionary studies and clinical applications.

Measuring short electron bunch lengths using coherent smith-purcell radiation

DOEpatents

Nguyen, Dinh C.

1999-01-01

A method is provided for directly determining the length of sub-picosecond electron bunches. A metallic grating is formed with a groove spacing greater than a length expected for the electron bunches. The electron bunches are passed over the metallic grating to generate coherent and incoherent Smith-Purcell radiation. The angular distribution of the coherent Smith-Purcell radiation is then mapped to directly deduce the length of the electron bunches.

Measuring short electron bunch lengths using coherent Smith-Purcell radiation

DOEpatents

Nguyen, D.C.

1999-03-30

A method is provided for directly determining the length of sub-picosecond electron bunches. A metallic grating is formed with a groove spacing greater than a length expected for the electron bunches. The electron bunches are passed over the metallic grating to generate coherent and incoherent Smith-Purcell radiation. The angular distribution of the coherent Smith-Purcell radiation is then mapped to directly deduce the length of the electron bunches. 8 figs.

Osteopontin-a Alters Glucose Homeostasis in Anchorage-Independent Breast Cancer Cells*

PubMed Central

Wang, Bo; Kennedy, Michael A.; Weber, Georg F.

2013-01-01

Invasive breast tumor cells generate three splice variants of the metastasis gene osteopontin, while non-invasive breast cells express only the unspliced form or no osteopontin at all. One role for osteopontin in tumor progression is the support of anchorage-independence. Here we show that the full-length gene product, osteopontin-a, induces a gene expression profile that is associated with tissue remodeling and directed movement/sprouting. This occurs via signals through STAT1 and STAT3 to snglycero-3-phosphocholine. Osteopontin-a upregulates the levels of glucose in breast cancer cells, likely through STAT3 and its transcriptional targets apolipoprotein D and IGFBP5. The splice variants osteopontin-a and osteopontin-c may synergize, with each form activating signal transduction pathways that are distinct from the other. The elevated glucose is used by osteopontin-c dependent signals to generate chemical energy (Shi et al. manuscript submitted). The splice variant-specific metabolic effects of osteopontin add a novel aspect to the pro-metastatic functions of this molecule. PMID:24157812

Method for computationally efficient design of dielectric laser accelerator structures

DOE PAGES

Hughes, Tyler; Veronis, Georgios; Wootton, Kent P.; ...

2017-06-22

Here, dielectric microstructures have generated much interest in recent years as a means of accelerating charged particles when powered by solid state lasers. The acceleration gradient (or particle energy gain per unit length) is an important figure of merit. To design structures with high acceleration gradients, we explore the adjoint variable method, a highly efficient technique used to compute the sensitivity of an objective with respect to a large number of parameters. With this formalism, the sensitivity of the acceleration gradient of a dielectric structure with respect to its entire spatial permittivity distribution is calculated by the use of onlymore » two full-field electromagnetic simulations, the original and ‘adjoint’. The adjoint simulation corresponds physically to the reciprocal situation of a point charge moving through the accelerator gap and radiating. Using this formalism, we perform numerical optimizations aimed at maximizing acceleration gradients, which generate fabricable structures of greatly improved performance in comparison to previously examined geometries.« less

Multiport backside-illuminated CCD imagers for high-frame-rate camera applications

NASA Astrophysics Data System (ADS)

Levine, Peter A.; Sauer, Donald J.; Hseuh, Fu-Lung; Shallcross, Frank V.; Taylor, Gordon C.; Meray, Grazyna M.; Tower, John R.; Harrison, Lorna J.; Lawler, William B.

1994-05-01

Two multiport, second-generation CCD imager designs have been fabricated and successfully tested. They are a 16-port 512 X 512 array and a 32-port 1024 X 1024 array. Both designs are back illuminated, have on-chip CDS, lateral blooming control, and use a split vertical frame transfer architecture with full frame storage. The 512 X 512 device has been operated at rates over 800 frames per second. The 1024 X 1024 device has been operated at rates over 300 frames per second. The major changes incorporated in the second-generation design are, reduction in gate length in the output area to give improved high-clock-rate performance, modified on-chip CDS circuitry for reduced noise, and optimized implants to improve performance of blooming control at lower clock amplitude. This paper discusses the imager design improvements and presents measured performance results at high and moderate frame rates. The design and performance of three moderate frame rate cameras are discussed.

Generation of PCV2 in PK15 cells transfected with recombinant baculovirus containing a 1.1 copy of the PCV2 genome.

PubMed

Cai, Jie; Xie, Xiaohong; Hu, Yi; Zhan, Yang; Yu, Wanting; Wang, Aibing; Wang, Naidong

2017-06-01

Porcine circovirus associated diseases (PCVAD) caused by PCV2 are responsible for severe economic losses in the swine industry. The mechanism of PCV2 replication has not been fully elucidated yet. PCV2 may be successfully rescued by means of either an infectious DNA clone containing the full length of the viral genomic DNA, or from PCV2-infected clinical tissues in PK15 cell culture. However, viruses harvested by both methods have low titres. In this study, PCV2 was prepared with a higher titre from PK15 cells infected by recombinant baculoviruses containing 1PCV2 (one stem-loop structure) or 1.1PCV2 (two stem-loop structure) genomic DNA copy. In addition, infectious DNA clones containing two stem-loop structures in either plasmid or baculovirus backbones are capable of generating a higher virus titre than the DNA clones with only one copy of stem-loop structure.

Stabilization of the μ-opioid receptor by truncated single transmembrane splice variants through a chaperone-like action.

PubMed

Xu, Jin; Xu, Ming; Brown, Taylor; Rossi, Grace C; Hurd, Yasmin L; Inturrisi, Charles E; Pasternak, Gavril W; Pan, Ying-Xian

2013-07-19

The μ-opioid receptor gene, OPRM1, undergoes extensive alternative pre-mRNA splicing, as illustrated by the identification of an array of splice variants generated by both 5' and 3' alternative splicing. The current study reports the identification of another set of splice variants conserved across species that are generated through exon skipping or insertion that encodes proteins containing only a single transmembrane (TM) domain. Using a Tet-Off system, we demonstrated that the truncated single TM variants can dimerize with the full-length 7-TM μ-opioid receptor (MOR-1) in the endoplasmic reticulum, leading to increased expression of MOR-1 at the protein level by a chaperone-like function that minimizes endoplasmic reticulum-associated degradation. In vivo antisense studies suggested that the single TM variants play an important role in morphine analgesia, presumably through modulation of receptor expression levels. Our studies suggest the functional roles of truncated receptors in other G protein-coupled receptor families.

Isoform Sequencing and State-of-Art Applications for Unravelling Complexity of Plant Transcriptomes

PubMed Central

An, Dong; Li, Changsheng; Humbeck, Klaus

2018-01-01

Single-molecule real-time (SMRT) sequencing developed by PacBio, also called third-generation sequencing (TGS), offers longer reads than the second-generation sequencing (SGS). Given its ability to obtain full-length transcripts without assembly, isoform sequencing (Iso-Seq) of transcriptomes by PacBio is advantageous for genome annotation, identification of novel genes and isoforms, as well as the discovery of long non-coding RNA (lncRNA). In addition, Iso-Seq gives access to the direct detection of alternative splicing, alternative polyadenylation (APA), gene fusion, and DNA modifications. Such applications of Iso-Seq facilitate the understanding of gene structure, post-transcriptional regulatory networks, and subsequently proteomic diversity. In this review, we summarize its applications in plant transcriptome study, specifically pointing out challenges associated with each step in the experimental design and highlight the development of bioinformatic pipelines. We aim to provide the community with an integrative overview and a comprehensive guidance to Iso-Seq, and thus to promote its applications in plant research. PMID:29346292

De novo assembly and next-generation sequencing to analyse full-length gene variants from codon-barcoded libraries.

PubMed

Cho, Namjin; Hwang, Byungjin; Yoon, Jung-ki; Park, Sangun; Lee, Joongoo; Seo, Han Na; Lee, Jeewon; Huh, Sunghoon; Chung, Jinsoo; Bang, Duhee

2015-09-21

Interpreting epistatic interactions is crucial for understanding evolutionary dynamics of complex genetic systems and unveiling structure and function of genetic pathways. Although high resolution mapping of en masse variant libraries renders molecular biologists to address genotype-phenotype relationships, long-read sequencing technology remains indispensable to assess functional relationship between mutations that lie far apart. Here, we introduce JigsawSeq for multiplexed sequence identification of pooled gene variant libraries by combining a codon-based molecular barcoding strategy and de novo assembly of short-read data. We first validate JigsawSeq on small sub-pools and observed high precision and recall at various experimental settings. With extensive simulations, we then apply JigsawSeq to large-scale gene variant libraries to show that our method can be reliably scaled using next-generation sequencing. JigsawSeq may serve as a rapid screening tool for functional genomics and offer the opportunity to explore evolutionary trajectories of protein variants.

Probing cooperative force generation in collective cancer invasion

NASA Astrophysics Data System (ADS)

Alobaidi, Amani A.; Xu, Yaopengxiao; Chen, Shaohua; Jiao, Yang; Sun, Bo

2017-08-01

Collective cellular dynamics in the three-dimensional extracellular matrix (ECM) plays a crucial role in many physiological processes such as cancer invasion. Both chemical and mechanical signaling support cell-cell communications on a variety of length scales, leading to collective migratory behaviors. Here we conduct experiments using 3D in vitro tumor models and develop a phenomenological model in order to probe the cooperativity of force generation in the collective invasion of breast cancer cells. In our model, cell-cell communication is characterized by a single parameter that quantifies the correlation length of cellular migration cycles. We devise a stochastic reconstruction method to generate realizations of cell colonies with specific contraction phase correlation functions and correlation length a. We find that as a increases, the characteristic size of regions containing cells with similar contraction phases grows. For small a values, the large fluctuations in individual cell contraction phases smooth out the temporal fluctuations in the time-dependent deformation field in the ECM. For large a values, the periodicity of an individual cell contraction cycle is clearly manifested in the temporal variation of the overall deformation field in the ECM. Through quantitative comparisons of the simulated and experimentally measured deformation fields, we find that the correlation length for collective force generation in the breast cancer diskoid in geometrically micropatterned ECM (DIGME) system is a≈ 25~μ \\text{m} , which is roughly twice the linear size of a single cell. One possible mechanism for this intermediate cell correlation length is the fiber-mediated stress propagation in the 3D ECM network in the DIGME system.

West Nile virus (WNV) genome RNAs with up to three adjacent mutations that disrupt long distance 5'-3' cyclization sequence basepairs are viable

DOE Office of Scientific and Technical Information (OSTI.GOV)

Basu, Mausumi; Brinton, Margo A., E-mail: mbrinton@gsu.ed

2011-03-30

Mosquito-borne flavivirus genomes contain conserved 5' and 3' cyclization sequences (CYC) that facilitate long distance RNA-RNA interactions. In previous studies, flavivirus replicon RNA replication was completely inhibited by single or multiple mismatching CYC nt substitutions. In the present study, full-length WNV genomes with one, two or three mismatching CYC substitutions showed reduced replication efficiencies but were viable and generated revertants with increased replication efficiency. Several different three adjacent mismatching CYC substitution mutant RNAs were rescued by a second site mutation that created an additional basepair (nts 147-10913) on the internal genomic side of the 5'-3' CYC. The finding that full-lengthmore » genomes with up to three mismatching CYC mutations are viable and can be rescued by a single nt spontaneous mutation indicates that more than three adjacent CYC basepair substitutions would be required to increase the safety of vaccine genomes by creating mismatches in inter-genomic recombinants.« less

Construction and characterization of HIV type 1 CRF07_BC infectious molecular clone from men who have sex with men.

PubMed

Jiang, Yan-Ling; Bai, Wen-Wei; Qu, Fan-Wei; Ma, Hua; Jiang, Run-Sheng; Shen, Bao-Sheng

2016-03-01

This study aimed to investigate the biological characterization of HIV type 1 (HIV-1) CRF07_BC infection among men who have sex with men (MSM). From November 2011 to November 2013, a total of 66 blood samples were collected from MSM with acute HIV-1 infection with CRF07_BC subgroup strains. Deletion in the gag p6 region was detected by sequence alignment and comparative analysis. Peripheral blood mononuclear cells (PBMCs) of HNXX1301-1307 samples were separated by density gradient centrifugation. Nested polymerase chain reaction (nPCR) was used to amplify the viral DNA. The near full-length HIV-1 DNA products were ligated to the long terminal repeat (LTR) vector plasmid (07BCLTR) to construct a full-length HIV clone. The molecular clone was transfected into HEK-293T cells, TZM-b1 cells and patients' PBMCs. The pregenome of an infectious molecular clone of HIV-1 (pNL4-3) was amplified, and a subclone with CRF07_BC was developed to construct the full-length chimeric molecular clone pNL4-3/07BCLTR. Detection of p24 antigen and luciferase activity was used to measure the in vitro infectivity of pNL4-3/07BCLTR. Among the 66 MSM patients infected with CRF07_BC strains, deletion mutations of the Gag P6 proteins were found in 7 of 18CRF07_BC strains; deletion mutations of 2-13 amino acids in different regions were discovered in 6 strains; and the remaining 42 strains did not show deletions. Seven strains with amino acids deficiency in the P6 protein accounted for 27% of all strains and 75% of all deletion genotype strains. A total of 186 full-length molecular clones of CRF07_BC were constructed. There were 5, 9, 10 and 11 clones of HNXX1302, HNXX1304, HNXX1305 and HNXX1306 that resulted in p24-positive supernatant when transfected into HEK-293T cells. Full-length clones of HNXX1302, HNXX1304, HNXX1305 and HNXX1306 showed slight infection in the transfected TZM-b1 cells, as judged by the fluorescence values of TZM-b1 cells 48h post-transfection. However, we were unable to transfect the patients' PMBCs with the above four clones. The phylogenetic tree of the C2V3 segment of the Env gene showed that a significant gene cluster was formed by all of the chimeric full-length HNXX1306 clones, and the bootstrap value for this cluster was 97.5%. Patients' PBMCs could be infected by 1306N6, 1306N13 and 1306N22 chimeric full-length clones. The CRF07_BC subtype (6889-7407 nucleotide residues of HXB2) is one of the most prevalent epidemic HIV-1 virus strains among the MSM population. The full-length chimeric molecular clone pNL4-3/07BCLTR may significantly improve the in vitro infectivity of the CRF07_BC strain. Copyright © 2016 Elsevier B.V. All rights reserved.

Development of a perturbation generator for vortex stability studies

NASA Technical Reports Server (NTRS)

Riester, J. E.; Ash, Robert L.

1991-01-01

Theory predicts vortex instability when subjected to certain types of disturbances. It was desired to build a device which could introduce controlled velocity perturbations into a trailing line vortex in order to study the effects on stability. A perturbation generator was designed and manufactured which can be attached to the centerbody of an airfoil type vortex generator. Details of design tests and manufacturing of the perturbation generator are presented. The device produced controlled perturbation with frequencies in excess of 250 Hz. Preliminary testing and evaluation of the perturbation generator performance was conducted in a 4 inch cylindrical pipe. Observations of vortex shedding frequencies from a centerbody were measured. Further evaluation with the perturbation generator attached to the vortex generator in a 2 x 3 foot wind tunnel were also conducted. Hot-wire anemometry was used to confirm the perturbation generator's ability to introduce controlled frequency fluctuations. Comparison of the energy levels of the disturbances in the vortex core was made between locations 42 chord lengths and 15 chord lengths downstream.

BioRAT: extracting biological information from full-length papers.

PubMed

Corney, David P A; Buxton, Bernard F; Langdon, William B; Jones, David T

2004-11-22

Converting the vast quantity of free-format text found in journals into a concise, structured format makes the researcher's quest for information easier. Recently, several information extraction systems have been developed that attempt to simplify the retrieval and analysis of biological and medical data. Most of this work has used the abstract alone, owing to the convenience of access and the quality of data. Abstracts are generally available through central collections with easy direct access (e.g. PubMed). The full-text papers contain more information, but are distributed across many locations (e.g. publishers' web sites, journal web sites and local repositories), making access more difficult. In this paper, we present BioRAT, a new information extraction (IE) tool, specifically designed to perform biomedical IE, and which is able to locate and analyse both abstracts and full-length papers. BioRAT is a Biological Research Assistant for Text mining, and incorporates a document search ability with domain-specific IE. We show first, that BioRAT performs as well as existing systems, when applied to abstracts; and second, that significantly more information is available to BioRAT through the full-length papers than via the abstracts alone. Typically, less than half of the available information is extracted from the abstract, with the majority coming from the body of each paper. Overall, BioRAT recalled 20.31% of the target facts from the abstracts with 55.07% precision, and achieved 43.6% recall with 51.25% precision on full-length papers.

The new albumin-free recombinant factor VIII concentrates for treatment of hemophilia: do they represent an actual incremental improvement?

PubMed

Josephson, Cassandra D; Abshire, Thomas

2004-07-01

The goal of eliminating the low levels of infectious disease risk from hemophilia treatment has resulted in the development of multiple generations of recombinant factor VIII (rFVIII) products. The ideal product should be devoid of human and animal proteins, which may transmit infectious agents. These products should also maintain molecular integrity, hemostatic efficacy, similar immunogenicity, and acceptable side effect profiles as compared to plasma-derived factor VIII. Currently available first-, second-, and third-generation rFVIII products include Recombinate; Kogenate FS/Helixate FS and ReFacto; and Advate, respectively. During the evolution of rFVIII products, either full-length or B-domain-deleted factor VIII were transfected into immortalized cell lines. The B-domain-deleted product, ReFacto, has resulted in an additional method to monitor factor VIII levels. The third-generation products offer the theoretical advantage of being produced without human and/or animal proteins. Upon initial introduction into the marketplace, the newer products have a higher cost. However, when analyzing historical trends, the prices of these products are almost equivalent to first-generation products within 3 years of licensure. Thus, the initial cost of the product may be a minimal issue in the medical decision process when selecting rFVIII replacement therapy.

Analysis of DNA Methylation of Gracilariopsis lemaneiformis Under Temperature Stress Using the Methylation Sensitive Amplification Polymorphism (MSAP) Technique

NASA Astrophysics Data System (ADS)

Peng, Chong; Sui, Zhenghong; Zhou, Wei; Hu, Yiyi; Mi, Ping; Jiang, Minjie; Li, Xiaodong; Ruan, Xudong

2018-06-01

Gracilariopsis lemaneiformis is an economically important agarophyte, which contains high quality gel and shows a high growth rate. Wild population of G. lemaneiformis displayed resident divergence, though with a low genetic diversity as was revealed by amplified fragment length polymorphism (AFLP) and simple sequence repeat (SSR) analyses. In addition, different strains of G. lemaneiformis are diverse in morphology. The highly inconsistence between genetic background and physiological characteristics recommends strongly to the regulation at epigenetic level. In this study, the DNA methylation change in G. lemaneiformis among different generation branches and under different temperature stresses was assessed using methylation sensitive amplified polymorphism (MSAP) technique. It was shown that DNA methylation level among different generation branches was diverse. The full and total methylated DNA level was the lowest in the second generation branch and the highest in the third generation. The total methylation level was 61.11%, 60.88% and 64.12% at 15°C, 22°C and 26°C, respectively. Compared with the control group (22°C), the fully methylated and totally methylated ratios were increased in both experiment groups (15°C and 26°C). All of the cytosine methylation/demethylation transform (CMDT) was further analyzed. High temperature treatment could induce more CMDT than low temperature treatment did.

Subcellular distribution of human RDM1 protein isoforms and their nucleolar accumulation in response to heat shock and proteotoxic stress.

PubMed

Messaoudi, Lydia; Yang, Yun-Gui; Kinomura, Aiko; Stavreva, Diana A; Yan, Gonghong; Bortolin-Cavaillé, Marie-Line; Arakawa, Hiroshi; Buerstedde, Jean-Marie; Hainaut, Pierre; Cavaillé, Jérome; Takata, Minoru; Van Dyck, Eric

2007-01-01

The RDM1 gene encodes a RNA recognition motif (RRM)-containing protein involved in the cellular response to the anti-cancer drug cisplatin in vertebrates. We previously reported a cDNA encoding the full-length human RDM1 protein. Here, we describe the identification of 11 human cDNAs encoding RDM1 protein isoforms. This repertoire is generated by alternative pre-mRNA splicing and differential usage of two translational start sites, resulting in proteins with long or short N-terminus and a great diversity in the exonic composition of their C-terminus. By using tagged proteins and fluorescent microscopy, we examined the subcellular distribution of full-length RDM1 (renamed RDM1alpha), and other RDM1 isoforms. We show that RDM1alpha undergoes subcellular redistribution and nucleolar accumulation in response to proteotoxic stress and mild heat shock. In unstressed cells, the long N-terminal isoforms displayed distinct subcellular distribution patterns, ranging from a predominantly cytoplasmic to almost exclusive nuclear localization, suggesting functional differences among the RDM1 proteins. However, all isoforms underwent stress-induced nucleolar accumulation. We identified nuclear and nucleolar localization determinants as well as domains conferring cytoplasmic retention to the RDM1 proteins. Finally, RDM1 null chicken DT40 cells displayed an increased sensitivity to heat shock, compared to wild-type (wt) cells, suggesting a function for RDM1 in the heat-shock response.

Pretest analysis of Semiscale Mod-3 baseline test S-07-8 and S-07-9

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fineman, C.P.; Steiner, J.L.; Snider, D.M.

This document contains a pretest analysis of the Semiscale Mod-3 system thermal-hydraulic response for the second and third integral tests in Test Series 7 (Tests S-07-8 and S-07-9). Test Series 7 is the first test series to be conducted with the Semiscale Mod-3 system. The design of the Mod-3 system includes an improved representation of certain portions of a pressurized water reactor (PWR) when compared to the previously operated Semiscale Mod-1 system. The improvements include a new vessel which contains a full length (3.66 m) core, a full length upper plenum and upper head, and an external downcomer. An activemore » pump and active steam generator scaled to their pressurized water reactor (PWR) counterparts have been added to the broken loop. The upper head design includes the capability to simulate emergency core coolant (ECC) injection into this region. Test Series 7 is divided into three groups of tests that emphasize the evaluation of the Mod-3 system performance during different phases of the loss-of-coolant experiment (LOCE) transient. The last test group, which includes Tests S-07-8 and S-07-9, will be used to evaluate the integral behavior of the system. The previous two test groups were used to evaluate the blowdown behavior and the reflood behavior of the system. 3 refs., 35 figs., 12 tabs.« less

AF4 and AF4N protein complexes: recruitment of P-TEFb kinase, their interactome and potential functions

PubMed Central

Scholz, Bastian; Kowarz, Eric; Rössler, Tanja; Ahmad, Khalil; Steinhilber, Dieter; Marschalek, Rolf

2015-01-01

AF4/AFF1 and AF5/AFF4 are the molecular backbone to assemble “super-elongation complexes” (SECs) that have two main functions: (1) control of transcriptional elongation by recruiting the positive transcription elongation factor b (P-TEFb = CyclinT1/CDK9) that is usually stored in inhibitory 7SK RNPs; (2) binding of different histone methyltransferases, like DOT1L, NSD1 and CARM1. This way, transcribed genes obtain specific histone signatures (e.g. H3K79me2/3, H3K36me2) to generate a transcriptional memory system. Here we addressed several questions: how is P-TEFb recruited into SEC, how is the AF4 interactome composed, and what is the function of the naturally occuring AF4N protein variant which exhibits only the first 360 amino acids of the AF4 full-length protein. Noteworthy, shorter protein variants are a specific feature of all AFF protein family members. Here, we demonstrate that full-length AF4 and AF4N are both catalyzing the transition of P-TEFb from 7SK RNP to their N-terminal domain. We have also mapped the protein-protein interaction network within both complexes. In addition, we have first evidence that the AF4N protein also recruits TFIIH and the tumor suppressor MEN1. This indicate that AF4N may have additional functions in transcriptional initiation and in MEN1-dependend transcriptional processes. PMID:26171280

Efficient and simpler method to construct normalized cDNA libraries with improved representations of full-length cDNAs

DOEpatents

Soares, Marcelo Bento; Bonaldo, Maria de Fatima

1998-01-01

This invention provides a method to normalize a cDNA library comprising: (a) constructing a directionally cloned library containing cDNA inserts wherein the insert is capable of being amplified by polymerase chain reaction; (b) converting a double-stranded cDNA library into single-stranded DNA circles; (c) generating single-stranded nucleic acid molecules complementary to the single-stranded DNA circles converted in step (b) by polymerase chain reaction with appropriate primers; (d) hybridizing the single-stranded DNA circles converted in step (b) with the complementary single-stranded nucleic acid molecules generated in step (c) to produce partial duplexes to an appropriate Cot; and (e) separating the unhybridized single-stranded DNA circles from the hybridized DNA circles, thereby generating a normalized cDNA library. This invention also provides a method to normalize a cDNA library wherein the generating of single-stranded nucleic acid molecules complementary to the single-stranded DNA circles converted in step (b) is by excising cDNA inserts from the double-stranded cDNA library; purifying the cDNA inserts from cloning vectors; and digesting the cDNA inserts with an exonuclease. This invention further provides a method to construct a subtractive cDNA library following the steps described above. This invention further provides normalized and/or subtractive cDNA libraries generated by the above methods.

Efficient and simpler method to construct normalized cDNA libraries with improved representations of full-length cDNAs

DOEpatents

Soares, M.B.; Fatima Bonaldo, M. de

1998-12-08

This invention provides a method to normalize a cDNA library comprising: (a) constructing a directionally cloned library containing cDNA inserts wherein the insert is capable of being amplified by polymerase chain reaction; (b) converting a double-stranded cDNA library into single-stranded DNA circles; (c) generating single-stranded nucleic acid molecules complementary to the single-stranded DNA circles converted in step (b) by polymerase chain reaction with appropriate primers; (d) hybridizing the single-stranded DNA circles converted in step (b) with the complementary single-stranded nucleic acid molecules generated in step (c) to produce partial duplexes to an appropriate Cot; and (e) separating the unhybridized single-stranded DNA circles from the hybridized DNA circles, thereby generating a normalized cDNA library. This invention also provides a method to normalize a cDNA library wherein the generating of single-stranded nucleic acid molecules complementary to the single-stranded DNA circles converted in step (b) is by excising cDNA inserts from the double-stranded cDNA library; purifying the cDNA inserts from cloning vectors; and digesting the cDNA inserts with an exonuclease. This invention further provides a method to construct a subtractive cDNA library following the steps described above. This invention further provides normalized and/or subtractive cDNA libraries generated by the above methods. 25 figs.

Architectural analysis and predicted functional capability of the human latissimus dorsi muscle.

PubMed

Gerling, Michael E; Brown, Stephen H M

2013-08-01

The latissimus dorsi is primarily considered a muscle with actions at the shoulder, despite its widespread attachments at the spine. There is some dispute regarding the potential contribution of this muscle to lumbar spine function. The architectural design of a muscle is one of the most accurate predictors of muscle function; however, detailed architectural data on the latissimus dorsi muscle are limited. Therefore, the aim of this study was to quantify the architectural properties of the latissimus dorsi muscle and model mechanical function in light of these new data. One latissimus dorsi muscle was removed from each of 12 human cadavers, separated into regions, and micro-dissected for quantification of fascicle length, sarcomere length, and physiological cross-sectional area. From these data, sarcomere length operating ranges were modelled to determine the force-length characteristics of latissimus dorsi across the spine and shoulder ranges of motion. The physiological cross-sectional area of latissimus dorsi was 5.6±0.5 cm2 and normalized fascicle length was 26.4±1.0 cm, indicating that this muscle is designed to produce a moderate amount of force over a large range of lengths. Measured sarcomere length in the post-mortem neutral spine posture was nearly optimal at 2.69±0.06 μm. Across spine range of motion, biomechanical modelling predicted latissimus dorsi acts across both the ascending and descending limbs of the force-length curve during lateral bend, and primarily at or near the plateau region (where maximum force generation is possible) during flexion/extension and axial twist. Across shoulder range of motion, latissimus dorsi acts primarily on the plateau region and descending limbs of the force length curve during both flexion/extension and abduction/adduction. These data provide novel insights into the ability of the latissimus dorsi muscle to generate force and change length throughout the spine and shoulder ranges of motion. In addition, these findings provide an improved understanding of the spine and shoulder positions at which the force-generating capacity of this muscle can become jeopardized, and consequently how this may affect its spine-stabilizing ability. © 2013 Anatomical Society.

Architectural analysis and predicted functional capability of the human latissimus dorsi muscle

PubMed Central

Gerling, Michael E; Brown, Stephen H M

2013-01-01

The latissimus dorsi is primarily considered a muscle with actions at the shoulder, despite its widespread attachments at the spine. There is some dispute regarding the potential contribution of this muscle to lumbar spine function. The architectural design of a muscle is one of the most accurate predictors of muscle function; however, detailed architectural data on the latissimus dorsi muscle are limited. Therefore, the aim of this study was to quantify the architectural properties of the latissimus dorsi muscle and model mechanical function in light of these new data. One latissimus dorsi muscle was removed from each of 12 human cadavers, separated into regions, and micro-dissected for quantification of fascicle length, sarcomere length, and physiological cross-sectional area. From these data, sarcomere length operating ranges were modelled to determine the force–length characteristics of latissimus dorsi across the spine and shoulder ranges of motion. The physiological cross-sectional area of latissimus dorsi was 5.6 ± 0.5 cm2 and normalized fascicle length was 26.4 ± 1.0 cm, indicating that this muscle is designed to produce a moderate amount of force over a large range of lengths. Measured sarcomere length in the post-mortem neutral spine posture was nearly optimal at 2.69 ± 0.06 μm. Across spine range of motion, biomechanical modelling predicted latissimus dorsi acts across both the ascending and descending limbs of the force–length curve during lateral bend, and primarily at or near the plateau region (where maximum force generation is possible) during flexion/extension and axial twist. Across shoulder range of motion, latissimus dorsi acts primarily on the plateau region and descending limbs of the force length curve during both flexion/extension and abduction/adduction. These data provide novel insights into the ability of the latissimus dorsi muscle to generate force and change length throughout the spine and shoulder ranges of motion. In addition, these findings provide an improved understanding of the spine and shoulder positions at which the force-generating capacity of this muscle can become jeopardized, and consequently how this may affect its spine-stabilizing ability. PMID:23758053

Reverse Genetics of Newcastle Disease Virus.

PubMed

Cardenas-Garcia, Stivalis; Afonso, Claudio L

2017-01-01

Reverse genetics allows for the generation of recombinant viruses or vectors used in functional studies, vaccine development, and gene therapy. This technique enables genetic manipulation and cloning of viral genomes, gene mutation through site-directed mutagenesis, along with gene insertion or deletion, among other studies. An in vitro infection-based system including the highly attenuated vaccinia virus Ankara strain expressing the T7 RNA polymerase from bacteriophage T7, with co-transfection of three helper plasmids and a full-length cDNA plasmid, was successfully developed to rescue genetically modified Newcastle disease viruses in 1999. In this chapter, the materials and the methods involved in rescuing Newcastle disease virus (NDV) from cDNA, utilizing site-directed mutagenesis and gene replacement techniques, are described in detail.

Peptide mediators of cholesterol efflux

DOEpatents

Bielicki, John K.; Johansson, Jan

2013-04-09

The present invention provides a family of non-naturally occurring polypeptides having cholesterol efflux activity that parallels that of full-length apolipoproteins (e.g., Apo AI and Apo E), and having high selectivity for ABAC1 that parallels that of full-length apolipoproteins. The invention also provides compositions comprising such polypeptides, methods of identifying, screening and synthesizing such polypeptides, and methods of treating, preventing or diagnosing diseases and disorders associated with dyslipidemia, hypercholesterolemia and inflammation.

Potent and selective mediators of cholesterol efflux

DOEpatents

Bielicki, John K; Johansson, Jan

2015-03-24

The present invention provides a family of non-naturally occurring polypeptides having cholesterol efflux activity that parallels that of full-length apolipoproteins (e.g., Apo AI and Apo E), and having high selectivity for ABAC1 that parallels that of full-length apolipoproteins. The invention also provides compositions comprising such polypeptides, methods of identifying, screening and synthesizing such polypeptides, and methods of treating, preventing or diagnosing diseases and disorders associated with dyslipidemia, hypercholesterolemia and inflammation.

Structure-based inhibitors of tau aggregation

NASA Astrophysics Data System (ADS)

Seidler, P. M.; Boyer, D. R.; Rodriguez, J. A.; Sawaya, M. R.; Cascio, D.; Murray, K.; Gonen, T.; Eisenberg, D. S.

2018-02-01

Aggregated tau protein is associated with over 20 neurological disorders, which include Alzheimer's disease. Previous work has shown that tau's sequence segments VQIINK and VQIVYK drive its aggregation, but inhibitors based on the structure of the VQIVYK segment only partially inhibit full-length tau aggregation and are ineffective at inhibiting seeding by full-length fibrils. Here we show that the VQIINK segment is the more powerful driver of tau aggregation. Two structures of this segment determined by the cryo-electron microscopy method micro-electron diffraction explain its dominant influence on tau aggregation. Of practical significance, the structures lead to the design of inhibitors that not only inhibit tau aggregation but also inhibit the ability of exogenous full-length tau fibrils to seed intracellular tau in HEK293 biosensor cells into amyloid. We also raise the possibility that the two VQIINK structures represent amyloid polymorphs of tau that may account for a subset of prion-like strains of tau.

Structure of the full-length TRPV2 channel by cryo-EM

NASA Astrophysics Data System (ADS)

Huynh, Kevin W.; Cohen, Matthew R.; Jiang, Jiansen; Samanta, Amrita; Lodowski, David T.; Zhou, Z. Hong; Moiseenkova-Bell, Vera Y.

2016-03-01

Transient receptor potential (TRP) proteins form a superfamily Ca2+-permeable cation channels regulated by a range of chemical and physical stimuli. Structural analysis of a `minimal' TRP vanilloid subtype 1 (TRPV1) elucidated a mechanism of channel activation by agonists through changes in its outer pore region. Though homologous to TRPV1, other TRPV channels (TRPV2-6) are insensitive to TRPV1 activators including heat and vanilloids. To further understand the structural basis of TRPV channel function, we determined the structure of full-length TRPV2 at ~5 Å resolution by cryo-electron microscopy. Like TRPV1, TRPV2 contains two constrictions, one each in the pore-forming upper and lower gates. The agonist-free full-length TRPV2 has wider upper and lower gates compared with closed and agonist-activated TRPV1. We propose these newly revealed TRPV2 structural features contribute to diversity of TRPV channels.

Structure of the full-length TRPV2 channel by cryo-EM

PubMed Central

Huynh, Kevin W.; Cohen, Matthew R.; Jiang, Jiansen; Samanta, Amrita; Lodowski, David T.; Zhou, Z. Hong; Moiseenkova-Bell, Vera Y.

2016-01-01

Transient receptor potential (TRP) proteins form a superfamily Ca2+-permeable cation channels regulated by a range of chemical and physical stimuli. Structural analysis of a ‘minimal' TRP vanilloid subtype 1 (TRPV1) elucidated a mechanism of channel activation by agonists through changes in its outer pore region. Though homologous to TRPV1, other TRPV channels (TRPV2–6) are insensitive to TRPV1 activators including heat and vanilloids. To further understand the structural basis of TRPV channel function, we determined the structure of full-length TRPV2 at ∼5 Å resolution by cryo-electron microscopy. Like TRPV1, TRPV2 contains two constrictions, one each in the pore-forming upper and lower gates. The agonist-free full-length TRPV2 has wider upper and lower gates compared with closed and agonist-activated TRPV1. We propose these newly revealed TRPV2 structural features contribute to diversity of TRPV channels. PMID:27021073

Structure of the full-length TRPV2 channel by cryo-EM.

PubMed

Huynh, Kevin W; Cohen, Matthew R; Jiang, Jiansen; Samanta, Amrita; Lodowski, David T; Zhou, Z Hong; Moiseenkova-Bell, Vera Y

2016-03-29

Transient receptor potential (TRP) proteins form a superfamily Ca(2+)-permeable cation channels regulated by a range of chemical and physical stimuli. Structural analysis of a 'minimal' TRP vanilloid subtype 1 (TRPV1) elucidated a mechanism of channel activation by agonists through changes in its outer pore region. Though homologous to TRPV1, other TRPV channels (TRPV2-6) are insensitive to TRPV1 activators including heat and vanilloids. To further understand the structural basis of TRPV channel function, we determined the structure of full-length TRPV2 at ∼5 Å resolution by cryo-electron microscopy. Like TRPV1, TRPV2 contains two constrictions, one each in the pore-forming upper and lower gates. The agonist-free full-length TRPV2 has wider upper and lower gates compared with closed and agonist-activated TRPV1. We propose these newly revealed TRPV2 structural features contribute to diversity of TRPV channels.

The effect of two different renal denervation strategies on blood pressure in resistant hypertension: Comparison of full-length versus proximal renal artery ablation.

PubMed

Chen, Weijie; Ling, Zhiyu; Du, Huaan; Song, Wenxin; Xu, Yanping; Liu, Zengzhang; Su, Li; Xiao, Peilin; Yuan, Yuelong; Lu, Jiayi; Zhang, Jianhong; Li, Zhifeng; Shao, Jiang; Zhong, Bin; Zhou, Bei; Woo, Kamsang; Yin, Yuehui

2016-11-01

Renal denervation (RDN) is used to manage blood pressure (BP) in patients with resistant hypertension (rHT), but effectiveness is still a concern, and key arterial portion for successful RDN is not clear. The aim of this study was to investigate the efficacy and safety of proximal versus full-length renal artery ablation in patients with resistant hypertension (rHT). Forty-seven patients with rHT were randomly assigned to receive full-length ablation (n = 23) or proximal ablation (n = 24) of the renal arteries. All lesions were treated with radiofrequency energy via a saline-irrigated catheter. Office BP was measured during 12 months of follow-up and ambulatory BP at baseline and 6 months (n = 15 in each group). Compared with full-length ablation, proximal ablation reduced the number of ablation points in both the right (6.1 ± 0.7 vs. 3.3 ± 0.6, P < 0.001) and the left renal arteries (6.2 ± 0.7 vs. 3.3 ± 0.8, P < 0.001), with significantly shorter RF delivery time (P < 0.001), but higher RF power (P = 0.011). Baseline office BPs was 179.4 ± 13.7/102.8 ± 9.4 mm Hg in the full-length group and 181.9 ± 12.8/103.5 ± 8.9 mm Hg in the proximal group (P > 0.5). Similar office BPs was reduced by -39.4 ± 11.5/-20.9 ± 7.1 mm Hg at 6 months and -38.2 ± 10.3/-21.5 ± 5.8 mm Hg at 12 months in the full-length group (P < 0.001), -42.0 ± 11.6/-21.4 ± 7.9 mm Hg at 6 months and -40.9 ± 10.3/-22.1 ± 5.6 mm Hg at 12 months in the proximal group (P < 0.001), and progressive BP reductions were observed over the 6 months (P < 0.001) in both groups. The drop in ambulatory 24-hr SBP and DBP were significantly less than the drop in office BP (P < 0.001). No renovascular or other adverse complications were observed. The results indicate that proximal RDN has a similar efficacy and safety profile compared with full-length RDN, and propose the proximal artery as the key portion for RDN. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Electrochemical cell apparatus having axially distributed entry of a fuel-spent fuel mixture transverse to the cell lengths

DOEpatents

Reichner, P.; Dollard, W.J.

1991-01-08

An electrochemical apparatus is made having a generator section containing axially elongated electrochemical cells, a fresh gaseous feed fuel inlet, a gaseous feed oxidant inlet, and at least one gaseous spent fuel exit channel, where the spent fuel exit channel passes from the generator chamber to combine with the fresh feed fuel inlet at a mixing apparatus, reformable fuel mixture channel passes through the length of the generator chamber and connects with the mixing apparatus, that channel containing entry ports within the generator chamber, where the axis of the ports is transverse to the fuel electrode surfaces, where a catalytic reforming material is distributed near the reformable fuel mixture entry ports. 2 figures.

Progression of growth in the external ear from birth to maturity: a 2-year follow-up study in India.

PubMed

Purkait, Ruma

2013-06-01

This study aimed to follow the growth dynamics of auricular dimensions from birth to the age of 18 years. The norms of dimensions at different ages, the peak growth period and the maturity age of the dimensions are essential information to Physicians for early clinical diagnosis or for deciding the optimal time for surgery to correct abnormalities. For this study, 2,147 children belonging to central Indian population were measured in at least three sequential sessions. Eight dimensions including the physiognomic length and width of the ear and its morphologic width; conchal length, width, and depth; and lobular length and width were measured using anthropometric technique. Three new dimensions (tragal length and height and maximum width of the antihelix) were introduced in the study. Three indices (auricular, conchal, and lobular) also were derived. Most dimensions exhibited very rapid growth during the first 3-6 months of infancy and thereafter proceeded at a slow pace until adulthood. The smaller dimensions (conchal depth, tragal height, and maximum width of the antihelix) increased continuously throughout the growth period. At birth, most of the dimensions were 52-76 % of their adult size, while tragal length and height were less than half their adult size. Unlike the other dimensions, the lobule length was smaller in males, probably due to the higher frequency of hypoplastic and bow-shaped lobules among them. The width dimensions matured earlier, at 5.6-11 years, whereas the maturity age of lengths varied from 12 to 16 years. The data generated in the current study will be useful to Physicians as a guideline in correcting auricular deformity and in constructing age progression charts of the external ear. Knowledge concerning the maturation age of the ear will help law enforcement authorities in deciding when to use it for establishing personal identification. This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

On the physics of electron ejection from laser-irradiated overdense plasmas

DOE Office of Scientific and Technical Information (OSTI.GOV)

Thévenet, M.; Vincenti, H.; Faure, J.

2016-06-15

Using 1D and 2D PIC simulations, we describe and model the backward ejection of electron bunches when a laser pulse reflects off an overdense plasma with a short density gradient on its front side. The dependence on the laser intensity and gradient scale length is studied. It is found that during each laser period, the incident laser pulse generates a large charge-separation field, or plasma capacitor, which accelerates an attosecond bunch of electrons toward vacuum. This process is maximized for short gradient scale lengths and collapses when the gradient scale length is comparable to the laser wavelength. We develop amore » model that reproduces the electron dynamics and the dependence on laser intensity and gradient scale length. This process is shown to be strongly linked with high harmonic generation via the Relativistic Oscillating Mirror mechanism.« less

Integration of deep transcriptome and proteome analyses reveals the components of alkaloid metabolism in opium poppy cell cultures

PubMed Central

2010-01-01

Background Papaver somniferum (opium poppy) is the source for several pharmaceutical benzylisoquinoline alkaloids including morphine, the codeine and sanguinarine. In response to treatment with a fungal elicitor, the biosynthesis and accumulation of sanguinarine is induced along with other plant defense responses in opium poppy cell cultures. The transcriptional induction of alkaloid metabolism in cultured cells provides an opportunity to identify components of this process via the integration of deep transcriptome and proteome databases generated using next-generation technologies. Results A cDNA library was prepared for opium poppy cell cultures treated with a fungal elicitor for 10 h. Using 454 GS-FLX Titanium pyrosequencing, 427,369 expressed sequence tags (ESTs) with an average length of 462 bp were generated. Assembly of these sequences yielded 93,723 unigenes, of which 23,753 were assigned Gene Ontology annotations. Transcripts encoding all known sanguinarine biosynthetic enzymes were identified in the EST database, 5 of which were represented among the 50 most abundant transcripts. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) of total protein extracts from cell cultures treated with a fungal elicitor for 50 h facilitated the identification of 1,004 proteins. Proteins were fractionated by one-dimensional SDS-PAGE and digested with trypsin prior to LC-MS/MS analysis. Query of an opium poppy-specific EST database substantially enhanced peptide identification. Eight out of 10 known sanguinarine biosynthetic enzymes and many relevant primary metabolic enzymes were represented in the peptide database. Conclusions The integration of deep transcriptome and proteome analyses provides an effective platform to catalogue the components of secondary metabolism, and to identify genes encoding uncharacterized enzymes. The establishment of corresponding transcript and protein databases generated by next-generation technologies in a system with a well-defined metabolite profile facilitates an improved linkage between genes, enzymes, and pathway components. The proteome database represents the most relevant alkaloid-producing enzymes, compared with the much deeper and more complete transcriptome library. The transcript database contained full-length mRNAs encoding most alkaloid biosynthetic enzymes, which is a key requirement for the functional characterization of novel gene candidates. PMID:21083930

Cloning, Assembly, and Modification of the Primary Human Cytomegalovirus Isolate Toledo by Yeast-Based Transformation-Associated Recombination.

PubMed

Vashee, Sanjay; Stockwell, Timothy B; Alperovich, Nina; Denisova, Evgeniya A; Gibson, Daniel G; Cady, Kyle C; Miller, Kristofer; Kannan, Krishna; Malouli, Daniel; Crawford, Lindsey B; Voorhies, Alexander A; Bruening, Eric; Caposio, Patrizia; Früh, Klaus

2017-01-01

Genetic engineering of cytomegalovirus (CMV) currently relies on generating a bacterial artificial chromosome (BAC) by introducing a bacterial origin of replication into the viral genome using in vivo recombination in virally infected tissue culture cells. However, this process is inefficient, results in adaptive mutations, and involves deletion of viral genes to avoid oversized genomes when inserting the BAC cassette. Moreover, BAC technology does not permit the simultaneous manipulation of multiple genome loci and cannot be used to construct synthetic genomes. To overcome these limitations, we adapted synthetic biology tools to clone CMV genomes in Saccharomyces cerevisiae . Using an early passage of the human CMV isolate Toledo, we first applied transformation-associated recombination (TAR) to clone 16 overlapping fragments covering the entire Toledo genome in Saccharomyces cerevisiae . Then, we assembled these fragments by TAR in a stepwise process until the entire genome was reconstituted in yeast. Since next-generation sequence analysis revealed that the low-passage-number isolate represented a mixture of parental and fibroblast-adapted genomes, we selectively modified individual DNA fragments of fibroblast-adapted Toledo (Toledo-F) and again used TAR assembly to recreate parental Toledo (Toledo-P). Linear, full-length HCMV genomes were transfected into human fibroblasts to recover virus. Unlike Toledo-F, Toledo-P displayed characteristics of primary isolates, including broad cellular tropism in vitro and the ability to establish latency and reactivation in humanized mice. Our novel strategy thus enables de novo cloning of CMV genomes, more-efficient genome-wide engineering, and the generation of viral genomes that are partially or completely derived from synthetic DNA. IMPORTANCE The genomes of large DNA viruses, such as human cytomegalovirus (HCMV), are difficult to manipulate using current genetic tools, and at this time, it is not possible to obtain, molecular clones of CMV without extensive tissue culture. To overcome these limitations, we used synthetic biology tools to capture genomic fragments from viral DNA and assemble full-length genomes in yeast. Using an early passage of the HCMV isolate Toledo containing a mixture of wild-type and tissue culture-adapted virus. we directly cloned the majority sequence and recreated the minority sequence by simultaneous modification of multiple genomic regions. Thus, our novel approach provides a paradigm to not only efficiently engineer HCMV and other large DNA viruses on a genome-wide scale but also facilitates the cloning and genetic manipulation of primary isolates and provides a pathway to generating entirely synthetic genomes.

Cloning, Assembly, and Modification of the Primary Human Cytomegalovirus Isolate Toledo by Yeast-Based Transformation-Associated Recombination

PubMed Central

Vashee, Sanjay; Stockwell, Timothy B.; Alperovich, Nina; Denisova, Evgeniya A.; Gibson, Daniel G.; Cady, Kyle C.; Miller, Kristofer; Kannan, Krishna; Malouli, Daniel; Crawford, Lindsey B.; Voorhies, Alexander A.; Bruening, Eric; Caposio, Patrizia

2017-01-01

ABSTRACT Genetic engineering of cytomegalovirus (CMV) currently relies on generating a bacterial artificial chromosome (BAC) by introducing a bacterial origin of replication into the viral genome using in vivo recombination in virally infected tissue culture cells. However, this process is inefficient, results in adaptive mutations, and involves deletion of viral genes to avoid oversized genomes when inserting the BAC cassette. Moreover, BAC technology does not permit the simultaneous manipulation of multiple genome loci and cannot be used to construct synthetic genomes. To overcome these limitations, we adapted synthetic biology tools to clone CMV genomes in Saccharomyces cerevisiae. Using an early passage of the human CMV isolate Toledo, we first applied transformation-associated recombination (TAR) to clone 16 overlapping fragments covering the entire Toledo genome in Saccharomyces cerevisiae. Then, we assembled these fragments by TAR in a stepwise process until the entire genome was reconstituted in yeast. Since next-generation sequence analysis revealed that the low-passage-number isolate represented a mixture of parental and fibroblast-adapted genomes, we selectively modified individual DNA fragments of fibroblast-adapted Toledo (Toledo-F) and again used TAR assembly to recreate parental Toledo (Toledo-P). Linear, full-length HCMV genomes were transfected into human fibroblasts to recover virus. Unlike Toledo-F, Toledo-P displayed characteristics of primary isolates, including broad cellular tropism in vitro and the ability to establish latency and reactivation in humanized mice. Our novel strategy thus enables de novo cloning of CMV genomes, more-efficient genome-wide engineering, and the generation of viral genomes that are partially or completely derived from synthetic DNA. IMPORTANCE The genomes of large DNA viruses, such as human cytomegalovirus (HCMV), are difficult to manipulate using current genetic tools, and at this time, it is not possible to obtain, molecular clones of CMV without extensive tissue culture. To overcome these limitations, we used synthetic biology tools to capture genomic fragments from viral DNA and assemble full-length genomes in yeast. Using an early passage of the HCMV isolate Toledo containing a mixture of wild-type and tissue culture-adapted virus. we directly cloned the majority sequence and recreated the minority sequence by simultaneous modification of multiple genomic regions. Thus, our novel approach provides a paradigm to not only efficiently engineer HCMV and other large DNA viruses on a genome-wide scale but also facilitates the cloning and genetic manipulation of primary isolates and provides a pathway to generating entirely synthetic genomes. PMID:28989973

Fundamental study of flow field generated by rotorcraft blades using wide-field shadowgraph

NASA Technical Reports Server (NTRS)

Parthasarathy, S. P.; Cho, Y. I.; Back, L. H.

1985-01-01

The vortex trajectory and vortex wake generated by helicopter rotors are visualized using a wide-field shadowgraph technique. Use of a retro-reflective Scotchlite screen makes it possible to investigate the flow field generated by full-scale rotors. Tip vortex trajectories are visible in shadowgraphs for a range of tip Mach number of 0.38 to 0.60. The effect of the angle of attack is substantial. At an angle of attack greater than 8 degrees, the visibility of the vortex core is significant even at relatively low tip Mach numbers. The theoretical analysis of the sensitivity is carried out for a rotating blade. This analysis demonstrates that the sensitivity decreases with increasing dimensionless core radius and increases with increasing tip Mach number. The threshold value of the sensitivity is found to be 0.0015, below which the vortex core is not visible and above which it is visible. The effect of the optical path length is also discussed. Based on this investigation, it is concluded that the application of this wide-field shadowgraph technique to a large wind tunnel test should be feasible. In addition, two simultaneous shadowgraph views would allow three-dimensional reconstruction of vortex trajectories.

Structure of the torque ring of the flagellar motor and the molecular basis for rotational switching.

PubMed

Lee, Lawrence K; Ginsburg, Michael A; Crovace, Claudia; Donohoe, Mhairi; Stock, Daniela

2010-08-19

The flagellar motor drives the rotation of flagellar filaments at hundreds of revolutions per second, efficiently propelling bacteria through viscous media. The motor uses the potential energy from an electrochemical gradient of cations across the cytoplasmic membrane to generate torque. A rapid switch from anticlockwise to clockwise rotation determines whether a bacterium runs smoothly forward or tumbles to change its trajectory. A protein called FliG forms a ring in the rotor of the flagellar motor that is involved in the generation of torque through an interaction with the cation-channel-forming stator subunit MotA. FliG has been suggested to adopt distinct conformations that induce switching but these structural changes and the molecular mechanism of switching are unknown. Here we report the molecular structure of the full-length FliG protein, identify conformational changes that are involved in rotational switching and uncover the structural basis for the formation of the FliG torque ring. This allows us to propose a model of the complete ring and switching mechanism in which conformational changes in FliG reverse the electrostatic charges involved in torque generation.

DNA polymerase ι functions in the generation of tandem mutations during somatic hypermutation of antibody genes.

PubMed

Maul, Robert W; MacCarthy, Thomas; Frank, Ekaterina G; Donigan, Katherine A; McLenigan, Mary P; Yang, William; Saribasak, Huseyin; Huston, Donald E; Lange, Sabine S; Woodgate, Roger; Gearhart, Patricia J

2016-08-22

DNA polymerase ι (Pol ι) is an attractive candidate for somatic hypermutation in antibody genes because of its low fidelity. To identify a role for Pol ι, we analyzed mutations in two strains of mice with deficiencies in the enzyme: 129 mice with negligible expression of truncated Pol ι, and knock-in mice that express full-length Pol ι that is catalytically inactive. Both strains had normal frequencies and spectra of mutations in the variable region, indicating that loss of Pol ι did not change overall mutagenesis. We next examined if Pol ι affected tandem mutations generated by another error-prone polymerase, Pol ζ. The frequency of contiguous mutations was analyzed using a novel computational model to determine if they occur during a single DNA transaction or during two independent events. Analyses of 2,000 mutations from both strains indicated that Pol ι-compromised mice lost the tandem signature, whereas C57BL/6 mice accumulated significant amounts of double mutations. The results support a model where Pol ι occasionally accesses the replication fork to generate a first mutation, and Pol ζ extends the mismatch with a second mutation. @2016.

DNA polymerase ι functions in the generation of tandem mutations during somatic hypermutation of antibody genes

PubMed Central

Donigan, Katherine A.; Huston, Donald E.; Lange, Sabine S.

2016-01-01

DNA polymerase ι (Pol ι) is an attractive candidate for somatic hypermutation in antibody genes because of its low fidelity. To identify a role for Pol ι, we analyzed mutations in two strains of mice with deficiencies in the enzyme: 129 mice with negligible expression of truncated Pol ι, and knock-in mice that express full-length Pol ι that is catalytically inactive. Both strains had normal frequencies and spectra of mutations in the variable region, indicating that loss of Pol ι did not change overall mutagenesis. We next examined if Pol ι affected tandem mutations generated by another error-prone polymerase, Pol ζ. The frequency of contiguous mutations was analyzed using a novel computational model to determine if they occur during a single DNA transaction or during two independent events. Analyses of 2,000 mutations from both strains indicated that Pol ι–compromised mice lost the tandem signature, whereas C57BL/6 mice accumulated significant amounts of double mutations. The results support a model where Pol ι occasionally accesses the replication fork to generate a first mutation, and Pol ζ extends the mismatch with a second mutation. PMID:27455952

Nearly arc-length tool path generation and tool radius compensation algorithm research in FTS turning

NASA Astrophysics Data System (ADS)

Zhao, Minghui; Zhao, Xuesen; Li, Zengqiang; Sun, Tao

2014-08-01

In the non-rotational symmetrical microstrcture surfaces generation using turning method with Fast Tool Servo(FTS), non-uniform distribution of the interpolation data points will lead to long processing cycle and poor surface quality. To improve this situation, nearly arc-length tool path generation algorithm is proposed, which generates tool tip trajectory points in nearly arc-length instead of the traditional interpolation rule of equal angle and adds tool radius compensation. All the interpolation points are equidistant in radial distribution because of the constant feeding speed in X slider, the high frequency tool radius compensation components are in both X direction and Z direction, which makes X slider difficult to follow the input orders due to its large mass. Newton iterative method is used to calculate the neighboring contour tangent point coordinate value with the interpolation point X position as initial value, in this way, the new Z coordinate value is gotten, and the high frequency motion components in X direction is decomposed into Z direction. Taking a typical microstructure with 4μm PV value for test, which is mixed with two 70μm wave length sine-waves, the max profile error at the angle of fifteen is less than 0.01μm turning by a diamond tool with big radius of 80μm. The sinusoidal grid is machined on a ultra-precision lathe succesfully, the wavelength is 70.2278μm the Ra value is 22.81nm evaluated by data points generated by filtering out the first five harmonics.

Direct generation of abruptly focusing vortex beams using a 3/2 radial phase-only pattern.

PubMed

Davis, Jeffrey A; Cottrell, Don M; Zinn, Jonathan M

2013-03-20

Abruptly focusing Airy beams have previously been generated using a radial cubic phase pattern that represents the Fourier transform of the Airy beam. The Fourier transform of this pattern is formed using a system length of 2f, where f is the focal length of the Fourier transform lens. In this work, we directly generate these abruptly focusing Airy beams using a 3/2 radial phase pattern encoded onto a liquid crystal display. The resulting optical system is much shorter. In addition, we can easily produce vortex patterns at the focal point of these beams. Experimental results match theoretical predictions.

Effect of Elastase-induced Emphysema on the Force-generating Ability of the Diaphragm

PubMed Central

Supinski, Gerald S.; Kelsen, Steven G.

1982-01-01

The effect of emphysema on the ability of the diaphragm to generate force was examined in costal diaphragm muscle strips from 10 Golden hamsters killed 18 mo after intratracheal injection of pancreatic elastase in a dose producing hyperinflation (mean total lung capacity [TLC] = 163% of control) and generalized panacinar emphysema. 13 saline-injected normal animals served as controls. The time course of isometric tension and the effect of alterations in muscle fiber and sarcomere length on the isometric tension (T) generated in response to tetanizing electrical stimuli (length-tension [L-T] relationship) were examined. Elastase administration caused an increase in diaphragm muscle thickness and reduction in the length of costal diaphragm muscle fibers measured in situ. Emphysema significantly increased the maximum tetanic tension as a result of hypertrophy. Maximal tension corrected for increases in muscle cross-sectional area (T/cm2), however, was the same in emphysematous (E) and control (C) animals. Emphysema also shifted the muscle fiber L-T curve of the diaphragm but not of a control muscle, the soleus, toward shorter lengths. In contrast to the effects of E on the diaphragm muscle fiber L-T curve, the sarcomere L-T curve was the same in E and C. Since the length at which tension was maximal correlated closely with sarcomere number (r = 0.94; P < 0.001) reduction in the number of sarcomeres in series in muscles from emphysematous animals appeared to explain the shift in the muscle fiber L-T curve. We conclude that in elastase-induced emphysema adaptive changes both in diaphragm cross-sectional area and sarcomere number augment the force-generating ability of the diaphragm. We speculate that changes in sarcomere number compensate for alterations in muscle fiber length resulting from chronic hyperinflation of the thorax, while diaphragmatic muscle hypertrophy represents a response to changes in respiratory load and/or diaphragm configuration (LaPlace relationship). Images PMID:6922866

Genetic diversity and natural selection of Plasmodium knowlesi merozoite surface protein 1 paralog gene in Malaysia.

PubMed

Ahmed, Md Atique; Fauzi, Muh; Han, Eun-Taek

2018-03-14

Human infections due to the monkey malaria parasite Plasmodium knowlesi is on the rise in most Southeast Asian countries specifically Malaysia. The C-terminal 19 kDa domain of PvMSP1P is a potential vaccine candidate, however, no study has been conducted in the orthologous gene of P. knowlesi. This study investigates level of polymorphisms, haplotypes and natural selection of full-length pkmsp1p in clinical samples from Malaysia. A total of 36 full-length pkmsp1p sequences along with the reference H-strain and 40 C-terminal pkmsp1p sequences from clinical isolates of Malaysia were downloaded from published genomes. Genetic diversity, polymorphism, haplotype and natural selection were determined using DnaSP 5.10 and MEGA 5.0 software. Genealogical relationships were determined using haplotype network tree in NETWORK software v5.0. Population genetic differentiation index (F ST ) and population structure of parasite was determined using Arlequin v3.5 and STRUCTURE v2.3.4 software. Comparison of 36 full-length pkmsp1p sequences along with the H-strain identified 339 SNPs (175 non-synonymous and 164 synonymous substitutions). The nucleotide diversity across the full-length gene was low compared to its ortholog pvmsp1p. The nucleotide diversity was higher toward the N-terminal domains (pkmsp1p-83 and 30) compared to the C-terminal domains (pkmsp1p-38, 33 and 19). Phylogenetic analysis of full-length genes identified 2 distinct clusters of P. knowlesi from Malaysian Borneo. The 40 pkmsp1p-19 sequences showed low polymorphisms with 16 polymorphisms leading to 18 haplotypes. In total there were 10 synonymous and 6 non-synonymous substitutions and 12 cysteine residues were intact within the two EGF domains. Evidence of strong purifying selection was observed within the full-length sequences as well in all the domains. Shared haplotypes of 40 pkmsp1p-19 were identified within Malaysian Borneo haplotypes. This study is the first to report on the genetic diversity and natural selection of pkmsp1p. A low level of genetic diversity and strong evidence of negative selection was detected and observed in all the domains of pkmsp1p of P. knowlesi indicating functional constrains. Shared haplotypes were identified within pkmsp1p-19 highlighting further evaluation using larger number of clinical samples from Malaysia.

[Complete genome sequencing and analyses of rabies viruses isolated from wild animals (Chinese Ferret-Badger) in Zhejiang province].

PubMed

Lei, Yong-Liang; Wang, Xiao-Guang; Liu, Fu-Ming; Chen, Xiu-Ying; Ye, Bi-Feng; Mei, Jian-Hua; Lan, Jin-Quan; Tang, Qing

2009-08-01

Based on sequencing the full-length genomes of two Chinese Ferret-Badger, we analyzed the properties of rabies viruses genetic variation in molecular level to get information on prevalence and variation of rabies viruses in Zhejiang, and to enrich the genome database of rabies viruses street strains isolated from Chinese wildlife. Overlapped fragments were amplified by RT-PCR and full-length genomes were assembled to analyze the nucleotide and deduced protein similarities and phylogenetic analyses of the N genes from Chinese Ferret-Badger, sika deer, vole, dog. Vaccine strains were then determined. The two full-length genomes were completely sequenced to find out that they had the same genetic structure with 11 923 nts including 58 nts-Leader, 1353 nts-NP, 894 nts-PP, 609 nts-MP, 1575 nts-GP, 6386 nts-LP, and 2, 5, 5 nts- intergenic regions (IGRs), 423 nts-Pseudogene-like sequence (Psi), 70 nts-Trailer. The two full-length genomes were in accordance with the properties of Rhabdoviridae Lyssa virus by blast and multi-sequence alignment. The nucleotide and amino acid sequences among Chinese strains had the highest similarity, especially among animals of the same species. Of the two full-length genomes, the similarity in amino acid level was dramatically higher than that in nucleotide level, so that the nucleotide mutations happened in these two genomes were most probably as synonymous mutations. Compared to the referenced rabies viruses, the lengths of the five protein coding regions did not show any changes or recombination, but only with a few-point mutations. It was evident that the five proteins appeared to be stable. The variation sites and types of the two ferret badgers genomes were similar to the referenced vaccine or street strains. The two strains were genotype 1 according to the multi-sequence and phylogenetic analyses, which possessing the distinct geographyphic characteristics of China. All the evidence suggested a cue that these two ferret badgers rabies viruses were likely to be street virus that already circulating in wildlife.

Evaluation of vector-primed cDNA library production from microgram quantities of total RNA.

PubMed

Kuo, Jonathan; Inman, Jason; Brownstein, Michael; Usdin, Ted B

2004-12-15

cDNA sequences are important for defining the coding region of genes, and full-length cDNA clones have proven to be useful for investigation of the function of gene products. We produced cDNA libraries containing 3.5-5 x 10(5) primary transformants, starting with 5 mug of total RNA prepared from mouse pituitary, adrenal, thymus, and pineal tissue, using a vector-primed cDNA synthesis method. Of approximately 1000 clones sequenced, approximately 20% contained the full open reading frames (ORFs) of known transcripts, based on the presence of the initiating methionine residue codon. The libraries were complex, with 94, 91, 83 and 55% of the clones from the thymus, adrenal, pineal and pituitary libraries, respectively, represented only once. Twenty-five full-length clones, not yet represented in the Mammalian Gene Collection, were identified. Thus, we have produced useful cDNA libraries for the isolation of full-length cDNA clones that are not yet available in the public domain, and demonstrated the utility of a simple method for making high-quality libraries from small amounts of starting material.

Construction of cDNA expression library of watermelon for isolation of ClWRKY1 transcription factors gene involved in resistance to Fusarium wilt.

PubMed

Yang, Bing-Yan; Huo, Xiu-Ai; Li, Peng-Fei; Wang, Cui-Xia; Duan, Hui-Jun

2014-08-01

Full-length cDNAs are very important for genome annotation and functional analysis of genes. The number of full-length cDNAs from watermelon remains limited. Here we report first the construction of a full-length enriched cDNA library from Fusarium wilt stressed watermelon (Citrullus lanatus Thunb.) cultivar PI296341 root tissues using the SMART method. The titer of primary cDNA library and amplified library was 2.21 x 10(6) and 2.0 x 10(10) pfu/ml, respectively and the rate of recombinant was above 85%. The size of insert fragment ranged from 0.3 to 2.0 kb. In this study, we first cloned a gene named ClWRKY1, which was 1981 bp long and encoded a protein consisting of 394 amino acids. It contained two characteristic WRKY domains and two zinc finger motifs. Quantitative real-time PCR showed that ClWRKY1 expression levels reached maximum level at 12 h after inoculation with Fusarium oxysporum f. sp. niveum. The full-length cDNA library of watermelon root tissues is not only essential for the cloning of genes which are known, but also an initial key for the screening and cloning of new genes that might be involved in resistance to Fusarium wilt.

Full-length silicone insoles versus ultrasound-guided corticosteroid injection in the management of plantar fasciitis: a randomized clinical trial.

PubMed

Yucel, Ufuk; Kucuksen, Sami; Cingoz, Havva T; Anliacik, Emel; Ozbek, Orhan; Salli, Ali; Ugurlu, Hatice

2013-12-01

Plantar fasciitis often leads to disability. Optimal treatment for this clinical condition is still unknown. To compare the effectiveness of wearing a full-length silicone insole with ultrasound-guided corticosteroid injection in the management of plantar fasciitis. Randomized clinical trial. Forty-two patients with chronic unilateral plantar fasciitis were allocated randomly to have an ultrasound-guided corticosteroid injection or wear a full-length silicone insole. Data were collected before the procedure and 1 month after. The primary outcome measures included first-step heel pain via Visual Analogue Scale and Heel Tenderness Index. Other outcome measures were the Foot and Ankle Outcome Score and ultrasonographic thickness of the plantar fascia. After 1 month, a significant improvement was shown in Visual Analogue Scale, Heel Tenderness Index, Foot and Ankle Outcome Score, and ultrasonographic thickness of plantar fascia in both groups. Visual Analogue Scale scores, Foot and Ankle Outcome Score pain, Foot and Ankle Outcome Score for activities of daily living, Foot and Ankle Outcome Score for sport and recreation function, and plantar fascia thickness were better in injection group than in insole group (p < 0.05). Although both ultrasound-guided corticosteroid injection and wearing a full-length silicone insole were effective in the conservative treatment of plantar fasciitis, we recommend the use of silicone insoles as a first line of treatment for persons with plantar fasciitis.

Nucleoplasmin-like domain of FKBP39 from Drosophila melanogaster forms a tetramer with partly disordered tentacle-like C-terminal segments

PubMed Central

Kozłowska, Małgorzata; Tarczewska, Aneta; Jakób, Michał; Bystranowska, Dominika; Taube, Michał; Kozak, Maciej; Czarnocki-Cieciura, Mariusz; Dziembowski, Andrzej; Orłowski, Marek; Tkocz, Katarzyna; Ożyhar, Andrzej

2017-01-01

Nucleoplasmins are a nuclear chaperone family defined by the presence of a highly conserved N-terminal core domain. X-ray crystallographic studies of isolated nucleoplasmin core domains revealed a β-propeller structure consisting of a set of five monomers that together form a stable pentamer. Recent studies on isolated N-terminal domains from Drosophila 39-kDa FK506-binding protein (FKBP39) and from other chromatin-associated proteins showed analogous, nucleoplasmin-like (NPL) pentameric structures. Here, we report that the NPL domain of the full-length FKBP39 does not form pentameric complexes. Multi-angle light scattering (MALS) and sedimentation equilibrium ultracentrifugation (SE AUC) analyses of the molecular mass of the full-length protein indicated that FKBP39 forms homotetrameric complexes. Molecular models reconstructed from small-angle X-ray scattering (SAXS) revealed that the NPL domain forms a stable, tetrameric core and that FK506-binding domains are linked to it by intrinsically disordered, flexible chains that form tentacle-like segments. Analyses of full-length FKBP39 and its isolated NPL domain suggested that the distal regions of the polypeptide chain influence and determine the quaternary conformation of the nucleoplasmin-like protein. These results provide new insights regarding the conserved structure of nucleoplasmin core domains and provide a potential explanation for the importance of the tetrameric structural organization of full-length nucleoplasmins. PMID:28074868

RT-PCR and sequence analysis of the full-length fusion protein of Canine Distemper Virus from domestic dogs.

PubMed

Romanutti, Carina; Gallo Calderón, Marina; Keller, Leticia; Mattion, Nora; La Torre, José

2016-02-01

During 2007-2014, 84 out of 236 (35.6%) samples from domestic dogs submitted to our laboratory for diagnostic purposes were positive for Canine Distemper Virus (CDV), as analyzed by RT-PCR amplification of a fragment of the nucleoprotein gene. Fifty-nine of them (70.2%) were from dogs that had been vaccinated against CDV. The full-length gene encoding the Fusion (F) protein of fifteen isolates was sequenced and compared with that of those of other CDVs, including wild-type and vaccine strains. Phylogenetic analysis using the F gene full-length sequences grouped all the Argentinean CDV strains in the SA2 clade. Sequence identity with the Onderstepoort vaccine strain was 89.0-90.6%, and the highest divergence was found in the 135 amino acids corresponding to the F protein signal-peptide, Fsp (64.4-66.7% identity). In contrast, this region was highly conserved among the local strains (94.1-100% identity). One extra putative N-glycosylation site was identified in the F gene of CDV Argentinean strains with respect to the vaccine strain. The present report is the first to analyze full-length F protein sequences of CDV strains circulating in Argentina, and contributes to the knowledge of molecular epidemiology of CDV, which may help in understanding future disease outbreaks. Copyright © 2015 Elsevier B.V. All rights reserved.

Hibiscus latent Fort Pierce virus in Brazil and synthesis of its biologically active full-length cDNA clone.

PubMed

Gao, Ruimin; Niu, Shengniao; Dai, Weifang; Kitajima, Elliot; Wong, Sek-Man

2016-10-01

A Brazilian isolate of Hibiscus latent Fort Pierce virus (HLFPV-BR) was firstly found in a hibiscus plant in Limeira, SP, Brazil. RACE PCR was carried out to obtain the full-length sequences of HLFPV-BR which is 6453 nucleotides and has more than 99.15 % of complete genomic RNA nucleotide sequence identity with that of HLFPV Japanese isolate. The genomic structure of HLFPV-BR is similar to other tobamoviruses. It includes a 5' untranslated region (UTR), followed by open reading frames encoding for a 128-kDa protein and a 188-kDa readthrough protein, a 38-kDa movement protein, 18-kDa coat protein, and a 3' UTR. Interestingly, the unique feature of poly(A) tract is also found within its 3'-UTR. Furthermore, from the total RNA extracted from the local lesions of HLFPV-BR-infected Chenopodium quinoa leaves, a biologically active, full-length cDNA clone encompassing the genome of HLFPV-BR was amplified and placed adjacent to a T7 RNA polymerase promoter. The capped in vitro transcripts from the cloned cDNA were infectious when mechanically inoculated into C. quinoa and Nicotiana benthamiana plants. This is the first report of the presence of an isolate of HLFPV in Brazil and the successful synthesis of a biologically active HLFPV-BR full-length cDNA clone.

Impact of Pre-Plasma on Electron Generation and Transport in Laser Plasma Interactions

NASA Astrophysics Data System (ADS)

Peebles, Jonathan Lee

Relativistic laser plasma interactions in conjunction with an underdense pre-plasma have been shown to generate a two temperature component electron spectrum. The lower temperature component described by "ponderomotive scaling'" is relatively well known and understood and is useful for applications such as the fast ignition inertial confinement fusion scheme. The higher energy electrons generated due to pre-plasma are denoted as "super-ponderomotive" electrons and facilitate interesting and useful applications. These include but are not limited to table top particle acceleration and generating high energy protons, x-rays and neutrons from secondary interactions. This dissertation describes experimental and particle-in-cell computational studies of the electron spectra produced from interactions between short pulse high intensity lasers and controlled pre-plasma conditions. Experiments were conducted at 3 laser labs: Texas Petawatt (University of Texas at Austin), Titan (Lawrence Livermore National Laboratory) and OMEGA-EP (University of Rochester). These lasers have different capabilities, and multiple experiments were carried out in order to fully understand super-ponderomotive electron generation and transport in the high intensity laser regime (I > 1018 W/cm2). In these experiments, an additional secondary long pulse beam was used to generate different scale lengths of "injected" pre-plasma while the pulse length and intensity of the short pulse beam were varied. The temperature and quantity of super-ponderomotive electrons were monitored with magnetic spectrometers and inferred via bremsstrahlung spectrometers while trajectory was estimated via Cu-Kalpha imaging. The experimental and simulation data show that super-ponderomotive electrons require pulse lengths of at least 450 fs to be accelerated and that higher intensity interactions generate large magnetic fields which cause severe deflection of the super-ponderomotive electrons. Laser incidence angle is shown to be extremely important in determining hot electron trajectory. Longer pulse length data taken on OMEGA-EP and Titan showed that super-ponderomotive electrons could be created without the need for an initial pre-plasma due to the underdense plasma created during the high intensity interaction alone.

Genetic analysis of a red tilapia (Oreochromis spp.) population undergoing three generations of selection for increased body weight at harvest.

PubMed

Hamzah, Azhar; Thoa, Ngo Phu; Nguyen, Nguyen Hong

2017-11-01

Quantitative genetic analysis was performed on 10,919 data records collected over three generations from the selection programme for increased body weight at harvest in red tilapia (Oreochromis spp.). They were offspring of 224 sires and 226 dams (50 sires and 60 dams per generation, on average). Linear mixed models were used to analyse body traits (weight, length, width and depth), whereas threshold generalised models assuming probit distribution were employed to examine genetic inheritance of survival rate, sexual maturity and body colour. The estimates of heritability for traits studied (body weight, standard length, body width, body depth, body colour, early sexual maturation and survival) across statistical models were moderate to high (0.13-0.45). Genetic correlations among body traits and survival were high and positive (0.68-0.96). Body length and width exhibited negative genetic correlations with body colour (- 0.47 to - 0.25). Sexual maturity was genetically correlated positively with measurements of body traits (weight and length). Direct and correlated genetic responses to selection were measured as estimated breeding values in each generation and expressed in genetic standard deviation units (σ G ). The cumulative improvement achieved for harvest body weight was 1.72 σ G after three generations or 12.5% per generation when the gain was expressed as a percentage of the base population. Selection for improved body weight also resulted in correlated increase in other body traits (length, width and depth) and survival rate (ranging from 0.25 to 0.81 genetic standard deviation units). Avoidance of black spot parent matings also improved the overall red colour of the selected population. It is concluded that the selective breeding programme for red tilapia has succeeded in achieving significant genetic improvement for a range of commercially important traits in this species, and the large genetic variation in body colour and survival also shows that there are prospects for future improvement of these traits in this population of red tilapia.

De novo assembly and transcriptome analysis of five major tissues of Jatropha curcas L. using GS FLX titanium platform of 454 pyrosequencing

PubMed Central

2011-01-01

Background Jatropha curcas L. is an important non-edible oilseed crop with promising future in biodiesel production. However, factors like oil yield, oil composition, toxic compounds in oil cake, pests and diseases limit its commercial potential. Well established genetic engineering methods using cloned genes could be used to address these limitations. Earlier, 10,983 unigenes from Sanger sequencing of ESTs, and 3,484 unique assembled transcripts from 454 pyrosequencing of uncloned cDNAs were reported. In order to expedite the process of gene discovery, we have undertaken 454 pyrosequencing of normalized cDNAs prepared from roots, mature leaves, flowers, developing seeds, and embryos of J. curcas. Results From 383,918 raw reads, we obtained 381,957 quality-filtered and trimmed reads that are suitable for the assembly of transcript sequences. De novo contig assembly of these reads generated 17,457 assembled transcripts (contigs) and 54,002 singletons. Average length of the assembled transcripts was 916 bp. About 30% of the transcripts were longer than 1000 bases, and the size of the longest transcript was 7,173 bases. BLASTX analysis revealed that 2,589 of these transcripts are full-length. The assembled transcripts were validated by RT-PCR analysis of 28 transcripts. The results showed that the transcripts were correctly assembled and represent actively expressed genes. KEGG pathway mapping showed that 2,320 transcripts are related to major biochemical pathways including the oil biosynthesis pathway. Overall, the current study reports 14,327 new assembled transcripts which included 2589 full-length transcripts and 27 transcripts that are directly involved in oil biosynthesis. Conclusion The large number of transcripts reported in the current study together with existing ESTs and transcript sequences will serve as an invaluable genetic resource for crop improvement in jatropha. Sequence information of those genes that are involved in oil biosynthesis could be used for metabolic engineering of jatropha to increase oil content, and to modify oil composition. PMID:21492485

De novo assembly and transcriptome analysis of five major tissues of Jatropha curcas L. using GS FLX titanium platform of 454 pyrosequencing.

PubMed

Natarajan, Purushothaman; Parani, Madasamy

2011-04-15

Jatropha curcas L. is an important non-edible oilseed crop with promising future in biodiesel production. However, factors like oil yield, oil composition, toxic compounds in oil cake, pests and diseases limit its commercial potential. Well established genetic engineering methods using cloned genes could be used to address these limitations. Earlier, 10,983 unigenes from Sanger sequencing of ESTs, and 3,484 unique assembled transcripts from 454 pyrosequencing of uncloned cDNAs were reported. In order to expedite the process of gene discovery, we have undertaken 454 pyrosequencing of normalized cDNAs prepared from roots, mature leaves, flowers, developing seeds, and embryos of J. curcas. From 383,918 raw reads, we obtained 381,957 quality-filtered and trimmed reads that are suitable for the assembly of transcript sequences. De novo contig assembly of these reads generated 17,457 assembled transcripts (contigs) and 54,002 singletons. Average length of the assembled transcripts was 916 bp. About 30% of the transcripts were longer than 1000 bases, and the size of the longest transcript was 7,173 bases. BLASTX analysis revealed that 2,589 of these transcripts are full-length. The assembled transcripts were validated by RT-PCR analysis of 28 transcripts. The results showed that the transcripts were correctly assembled and represent actively expressed genes. KEGG pathway mapping showed that 2,320 transcripts are related to major biochemical pathways including the oil biosynthesis pathway. Overall, the current study reports 14,327 new assembled transcripts which included 2589 full-length transcripts and 27 transcripts that are directly involved in oil biosynthesis. The large number of transcripts reported in the current study together with existing ESTs and transcript sequences will serve as an invaluable genetic resource for crop improvement in jatropha. Sequence information of those genes that are involved in oil biosynthesis could be used for metabolic engineering of jatropha to increase oil content, and to modify oil composition.

Application of High-Throughput Next-Generation Sequencing for HLA Typing on Buccal Extracted DNA: Results from over 10,000 Donor Recruitment Samples

PubMed Central

Nguyen, David; Valenzuela, Nicole; Takemura, Ping; Bolon, Yung-Tsi; Springer, Brianna; Saito, Katsuyuki; Zheng, Ying; Hague, Tim; Pasztor, Agnes; Horvath, Gyorgy; Rigo, Krisztina; Reed, Elaine F.; Zhang, Qiuheng

2016-01-01

Background Unambiguous HLA typing is important in hematopoietic stem cell transplantation (HSCT), HLA disease association studies, and solid organ transplantation. However, current molecular typing methods only interrogate the antigen recognition site (ARS) of HLA genes, resulting in many cis-trans ambiguities that require additional typing methods to resolve. Here we report high-resolution HLA typing of 10,063 National Marrow Donor Program (NMDP) registry donors using long-range PCR by next generation sequencing (NGS) approach on buccal swab DNA. Methods Multiplex long-range PCR primers amplified the full-length of HLA class I genes (A, B, C) from promotor to 3’ UTR. Class II genes (DRB1, DQB1) were amplified from exon 2 through part of exon 4. PCR amplicons were pooled and sheared using Covaris fragmentation. Library preparation was performed using the Illumina TruSeq Nano kit on the Beckman FX automated platform. Each sample was tagged with a unique barcode, followed by 2×250 bp paired-end sequencing on the Illumina MiSeq. HLA typing was assigned using Omixon Twin software that combines two independent computational algorithms to ensure high confidence in allele calling. Consensus sequence and typing results were reported in Histoimmunogenetics Markup Language (HML) format. All homozygous alleles were confirmed by Luminex SSO typing and exon novelties were confirmed by Sanger sequencing. Results Using this automated workflow, over 10,063 NMDP registry donors were successfully typed under high-resolution by NGS. Despite known challenges of nucleic acid degradation and low DNA concentration commonly associated with buccal-based specimens, 97.8% of samples were successfully amplified using long-range PCR. Among these, 98.2% were successfully reported by NGS, with an accuracy rate of 99.84% in an independent blind Quality Control audit performed by the NDMP. In this study, NGS-HLA typing identified 23 null alleles (0.023%), 92 rare alleles (0.091%) and 42 exon novelties (0.042%). Conclusion Long-range, unambiguous HLA genotyping is achievable on clinical buccal swab-extracted DNA. Importantly, full-length gene sequencing and the ability to curate full sequence data will permit future interrogation of the impact of introns, expanded exons, and other gene regulatory sequences on clinical outcomes in transplantation. PMID:27798706

Application of High-Throughput Next-Generation Sequencing for HLA Typing on Buccal Extracted DNA: Results from over 10,000 Donor Recruitment Samples.

PubMed

Yin, Yuxin; Lan, James H; Nguyen, David; Valenzuela, Nicole; Takemura, Ping; Bolon, Yung-Tsi; Springer, Brianna; Saito, Katsuyuki; Zheng, Ying; Hague, Tim; Pasztor, Agnes; Horvath, Gyorgy; Rigo, Krisztina; Reed, Elaine F; Zhang, Qiuheng

2016-01-01

Unambiguous HLA typing is important in hematopoietic stem cell transplantation (HSCT), HLA disease association studies, and solid organ transplantation. However, current molecular typing methods only interrogate the antigen recognition site (ARS) of HLA genes, resulting in many cis-trans ambiguities that require additional typing methods to resolve. Here we report high-resolution HLA typing of 10,063 National Marrow Donor Program (NMDP) registry donors using long-range PCR by next generation sequencing (NGS) approach on buccal swab DNA. Multiplex long-range PCR primers amplified the full-length of HLA class I genes (A, B, C) from promotor to 3' UTR. Class II genes (DRB1, DQB1) were amplified from exon 2 through part of exon 4. PCR amplicons were pooled and sheared using Covaris fragmentation. Library preparation was performed using the Illumina TruSeq Nano kit on the Beckman FX automated platform. Each sample was tagged with a unique barcode, followed by 2×250 bp paired-end sequencing on the Illumina MiSeq. HLA typing was assigned using Omixon Twin software that combines two independent computational algorithms to ensure high confidence in allele calling. Consensus sequence and typing results were reported in Histoimmunogenetics Markup Language (HML) format. All homozygous alleles were confirmed by Luminex SSO typing and exon novelties were confirmed by Sanger sequencing. Using this automated workflow, over 10,063 NMDP registry donors were successfully typed under high-resolution by NGS. Despite known challenges of nucleic acid degradation and low DNA concentration commonly associated with buccal-based specimens, 97.8% of samples were successfully amplified using long-range PCR. Among these, 98.2% were successfully reported by NGS, with an accuracy rate of 99.84% in an independent blind Quality Control audit performed by the NDMP. In this study, NGS-HLA typing identified 23 null alleles (0.023%), 92 rare alleles (0.091%) and 42 exon novelties (0.042%). Long-range, unambiguous HLA genotyping is achievable on clinical buccal swab-extracted DNA. Importantly, full-length gene sequencing and the ability to curate full sequence data will permit future interrogation of the impact of introns, expanded exons, and other gene regulatory sequences on clinical outcomes in transplantation.

Predictions of Transient Flame Lift-Off Length With Comparison to Single-Cylinder Optical Engine Experiments

DOE PAGES

Senecal, P. K.; Pomraning, E.; Anders, J. W.; ...

2014-05-28

A state-of-the-art, grid-convergent simulation methodology was applied to three-dimensional calculations of a single-cylinder optical engine. A mesh resolution study on a sector-based version of the engine geometry further verified the RANS-based cell size recommendations previously presented by Senecal et al. (“Grid Convergent Spray Models for Internal Combustion Engine CFD Simulations,” ASME Paper No. ICEF2012-92043). Convergence of cylinder pressure, flame lift-off length, and emissions was achieved for an adaptive mesh refinement cell size of 0.35 mm. Furthermore, full geometry simulations, using mesh settings derived from the grid convergence study, resulted in excellent agreement with measurements of cylinder pressure, heat release rate,more » and NOx emissions. On the other hand, the full geometry simulations indicated that the flame lift-off length is not converged at 0.35 mm for jets not aligned with the computational mesh. Further simulations suggested that the flame lift-off lengths for both the nonaligned and aligned jets appear to be converged at 0.175 mm. With this increased mesh resolution, both the trends and magnitudes in flame lift-off length were well predicted with the current simulation methodology. Good agreement between the overall predicted flame behavior and the available chemiluminescence measurements was also achieved. Our present study indicates that cell size requirements for accurate prediction of full geometry flame lift-off lengths may be stricter than those for global combustion behavior. This may be important when accurate soot predictions are required.« less

Predictions of Transient Flame Lift-Off Length With Comparison to Single-Cylinder Optical Engine Experiments

DOE Office of Scientific and Technical Information (OSTI.GOV)

Senecal, P. K.; Pomraning, E.; Anders, J. W.

A state-of-the-art, grid-convergent simulation methodology was applied to three-dimensional calculations of a single-cylinder optical engine. A mesh resolution study on a sector-based version of the engine geometry further verified the RANS-based cell size recommendations previously presented by Senecal et al. (“Grid Convergent Spray Models for Internal Combustion Engine CFD Simulations,” ASME Paper No. ICEF2012-92043). Convergence of cylinder pressure, flame lift-off length, and emissions was achieved for an adaptive mesh refinement cell size of 0.35 mm. Furthermore, full geometry simulations, using mesh settings derived from the grid convergence study, resulted in excellent agreement with measurements of cylinder pressure, heat release rate,more » and NOx emissions. On the other hand, the full geometry simulations indicated that the flame lift-off length is not converged at 0.35 mm for jets not aligned with the computational mesh. Further simulations suggested that the flame lift-off lengths for both the nonaligned and aligned jets appear to be converged at 0.175 mm. With this increased mesh resolution, both the trends and magnitudes in flame lift-off length were well predicted with the current simulation methodology. Good agreement between the overall predicted flame behavior and the available chemiluminescence measurements was also achieved. Our present study indicates that cell size requirements for accurate prediction of full geometry flame lift-off lengths may be stricter than those for global combustion behavior. This may be important when accurate soot predictions are required.« less

Role of anode length in a mather-type plasma focus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Beg, F.N.; Zakaullah, M.; Nisar, M.

In this paper, neutron emission from a 3 KJ Mather-type plasma focus is studied. Specifically, the behavior of system with the change in anode length is investigated. Anode lengths of high and low fluence anisotropy as well as for high neutron yield are identified. Experiment also suggest the possibility of ion beam generation leading to neutron production via beam-plasma interaction.

A high-throughput platform for population reformatting and mammalian expression of phage display libraries to enable functional screening as full-length IgG.

PubMed

Xiao, Xiaodong; Douthwaite, Julie A; Chen, Yan; Kemp, Ben; Kidd, Sara; Percival-Alwyn, Jennifer; Smith, Alison; Goode, Kate; Swerdlow, Bonnie; Lowe, David; Wu, Herren; Dall'Acqua, William F; Chowdhury, Partha S

Phage display antibody libraries are a rich resource for discovery of potential therapeutic antibodies. Single-chain variable fragment (scFv) libraries are the most common format due to the efficient display of scFv by phage particles and the ease by which soluble scFv antibodies can be expressed for high-throughput screening. Typically, a cascade of screening and triaging activities are performed, beginning with the assessment of large numbers of E. coli-expressed scFv, and progressing through additional assays with individual reformatting of the most promising scFv to full-length IgG. However, use of high-throughput screening of scFv for the discovery of full-length IgG is not ideal because of the differences between these molecules. Furthermore, the reformatting step represents a bottle neck in the process because each antibody has to be handled individually to preserve the unique VH and VL pairing. These problems could be resolved if populations of scFv could be reformatted to full-length IgG before screening without disrupting the variable region pairing. Here, we describe a novel strategy that allows the reformatting of diverse populations of scFv from phage selections to full-length IgG in a batch format. The reformatting process maintains the diversity and variable region pairing with high fidelity, and the resulted IgG pool enables high-throughput expression of IgG in mammalian cells and cell-based functional screening. The improved process led to the discovery of potent candidates that are comparable or better than those obtained by traditional methods. This strategy should also be readily applicable to Fab-based phage libraries. Our approach, Screening in Product Format (SiPF), represents a substantial improvement in the field of antibody discovery using phage display.

Detection of rearrangements and transcriptional up-regulation of ALK in FFPE lung cancer specimens using a novel, sensitive, quantitative reverse transcription polymerase chain reaction assay.

PubMed

Gruber, Kim; Horn, Heike; Kalla, Jörg; Fritz, Peter; Rosenwald, Andreas; Kohlhäufl, Martin; Friedel, Godehard; Schwab, Matthias; Ott, German; Kalla, Claudia

2014-03-01

The approved dual-color fluorescence in situ hybridization (FISH) test for the detection of anaplastic lymphoma receptor tyrosine kinase (ALK) gene rearrangements in non-small-cell lung cancer (NSCLC) is complex and represents a low-throughput assay difficult to use in daily diagnostic practice. We devised a sensitive and robust routine diagnostic test for the detection of rearrangements and transcriptional up-regulation of ALK. We developed a quantitative reverse transcription polymerase chain reaction (qRT-PCR) assay adapted to RNA isolated from routine formalin-fixed, paraffin-embedded material and applied it to 652 NSCLC specimens. The reliability of this technique to detect ALK dysregulation was shown by comparison with FISH and immunohistochemistry. qRT-PCR analysis detected unbalanced ALK expression indicative of a gene rearrangement in 24 (4.6%) and full-length ALK transcript expression in six (1.1%) of 523 interpretable tumors. Among 182 tumors simultaneously analyzed by FISH and qRT-PCR, the latter accurately typed 97% of 19 rearranged and 158 nonrearranged tumors and identified ALK deregulation in two cases with insufficient FISH. Six tumors expressing full-length ALK transcripts did not show rearrangements of the gene. Immunohistochemistry detected ALK protein overexpression in tumors with gene fusions and transcriptional up-regulation, but did not distinguish between the two. One case with full-length ALK expression carried a heterozygous point mutation (S1220Y) within the kinase domain potentially interfering with kinase activity and/or inhibitor binding. Our qRT-PCR assay reliably identifies and distinguishes ALK rearrangements and full-length transcript expression in formalin-fixed, paraffin-embedded material. It is an easy-to-perform, cost-effective, and high-throughput tool for the diagnosis of ALK activation. The expression of full-length ALK transcripts may be relevant for ALK inhibitor therapy in NSCLC.

An Evaluation of Two Methods for Generating Synthetic HL7 Segments Reflecting Real-World Health Information Exchange Transactions

PubMed Central

Mwogi, Thomas S.; Biondich, Paul G.; Grannis, Shaun J.

2014-01-01

Motivated by the need for readily available data for testing an open-source health information exchange platform, we developed and evaluated two methods for generating synthetic messages. The methods used HL7 version 2 messages obtained from the Indiana Network for Patient Care. Data from both methods were analyzed to assess how effectively the output reflected original ‘real-world’ data. The Markov Chain method (MCM) used an algorithm based on transitional probability matrix while the Music Box model (MBM) randomly selected messages of particular trigger type from the original data to generate new messages. The MBM was faster, generated shorter messages and exhibited less variation in message length. The MCM required more computational power, generated longer messages with more message length variability. Both methods exhibited adequate coverage, producing a high proportion of messages consistent with original messages. Both methods yielded similar rates of valid messages. PMID:25954458

The C-terminal domain of TRPV4 is essential for plasma membrane localization.

PubMed

Becker, Daniel; Müller, Margarethe; Leuner, Kristina; Jendrach, Marina

2008-02-01

Many members of the TRP superfamily oligomerize in the ER before trafficking to the plasma membrane. For membrane localization of the non-selective cation channel TRPV4 specific domains in the N-terminus are required, but the role of the C-terminus in the oligomerization and trafficking process has been not determined until now. Therefore, the localization of recombinant TRPV4 in two cell models was analyzed: HaCaT keratinocytes that express TRPV4 endogenously were compared to CHO cells that are devoid of endogenous TRPV4. When deletions were introduced in the C-terminal domain three states of TRPV4 localization were defined: a truncated TRPV4 protein of 855 amino acids was exported to the plasma membrane like the full-length channel (871 aa) and was also functional. Mutants with a length of 828 to 844 amino acids remained in the ER of CHO cells, but in HaCaT cells plasma membrane localization was partially rescued by oligomerization with endogenous TRPV4. This was confirmed by coexpression of recombinant full-length TRPV4 together with these deletion mutants, which resulted in an almost complete plasma membrane localization of both proteins and significant FRET in the plasma membrane and the ER. All deletions upstream of amino acid 828 resulted in total ER retention that could not rescued by coexpression with the full-length protein. However, these deletion mutants did not impair export of full-length TRPV4, implying that no oligomerization took place. These data indicate that the C-terminus of TRPV4 is required for oligomerization, which takes place in the ER and precedes plasma membrane trafficking.

Influenza A virus PB1-F2 protein prolongs viral shedding in chickens lengthening the transmission window

PubMed Central

James, Joe; Howard, Wendy; Iqbal, Munir; Nair, Venugopal K.; Barclay, Wendy S.

2016-01-01

Avian influenza is a significant economic burden on the poultry industry in geographical regions where it is enzootic. It also poses a public health concern when avian influenza subtypes infect humans, often with high mortality. Understanding viral genetic factors which positively contribute to influenza A virus (IAV) fitness – infectivity, spread and pathogenesis – is of great importance both for human and livestock health. PB1-F2 is a small accessory protein encoded by IAV and in mammalian hosts has been implicated in a wide range of functions that contribute to increased pathogenesis. In the avian host, the protein has been understudied despite high-level full-length conservation in avian IAV isolates, which is in contrast to the truncations of the PB1-F2 length frequently found in mammalian host isolates. Here we report that the presence of a full-length PB1-F2 protein, from a low pathogenicity H9N2 avian influenza virus, prolongs infectious virus shedding from directly inoculated chickens, thereby enhancing transmission of the virus by lengthening the transmission window to contact birds. As well as extending transmission, the presence of a full-length PB1-F2 suppresses pathogenicity evidenced by an increased minimum lethal dose in embryonated chicken eggs and increasing survival in directly infected birds when compared to a virus lacking an ORF for PB1-F2. We propose that there is a positive pressure to maintain a full-length functional PB1-F2 protein upon infection of avian hosts as it contributes to the effective transmission of IAV in the field. PMID:27558742

Influenza A virus PB1-F2 protein prolongs viral shedding in chickens lengthening the transmission window.

PubMed

James, Joe; Howard, Wendy; Iqbal, Munir; Nair, Venugopal K; Barclay, Wendy S; Shelton, Holly

2016-10-01

Avian influenza is a significant economic burden on the poultry industry in geographical regions where it is enzootic. It also poses a public health concern when avian influenza subtypes infect humans, often with high mortality. Understanding viral genetic factors which positively contribute to influenza A virus (IAV) fitness - infectivity, spread and pathogenesis - is of great importance both for human and livestock health. PB1-F2 is a small accessory protein encoded by IAV and in mammalian hosts has been implicated in a wide range of functions that contribute to increased pathogenesis. In the avian host, the protein has been understudied despite high-level full-length conservation in avian IAV isolates, which is in contrast to the truncations of the PB1-F2 length frequently found in mammalian host isolates. Here we report that the presence of a full-length PB1-F2 protein, from a low pathogenicity H9N2 avian influenza virus, prolongs infectious virus shedding from directly inoculated chickens, thereby enhancing transmission of the virus by lengthening the transmission window to contact birds. As well as extending transmission, the presence of a full-length PB1-F2 suppresses pathogenicity evidenced by an increased minimum lethal dose in embryonated chicken eggs and increasing survival in directly infected birds when compared to a virus lacking an ORF for PB1-F2. We propose that there is a positive pressure to maintain a full-length functional PB1-F2 protein upon infection of avian hosts as it contributes to the effective transmission of IAV in the field.

Processing, Assembly and Localization of a Bacillus anthracis Spore Protein

DTIC Science & Technology

2010-01-01

phage transduction, using the CP51 phage as described by Thorne (1968). All mutations were confirmed by PCR analysis (Supplementary Table S1). Protein...with End-It (Epicentre) and self-ligated, creating pKH-KSM4. The region between the T7 terminator and T7 promoter of pET23A (EMD Table 1. Strains and...represent full-length BxpA, we analysed the electrophoretic behaviour of a full-length, histidine-tagged and T7 -tagged version of BxpA overproduced in E

A third of patients treated at a tertiary-level surgical service could be treated at a secondary-level facility.

PubMed

Van Straten, S; Stannard, C; Bulabula, J; Boodhia, K; Paul, K; Leong, J; Klipin, M J

2017-08-25

South Africa (SA) has an overburdened public healthcare system. Some patients admitted to Charlotte Maxeke Johannesburg Academic Hospital (CMJAH), SA, may not require tertiary care, but the numbers and details are uncertain. Clinical research in SA is limited by scarce skills and limited access to data. To determine the proportion of and length of stay for secondary-, tertiary- and quaternary-level patients discharged from the Department of Surgery at CMJAH over 1 year. This is a retrospective analysis of electronic discharge (ED) summaries from the Department of Surgery at CMJAH between 1 April 2015 and 1 April 2016. An SQL query of the database generated a .csv file of all discharges with the following fields: database reference number, length of stay and level of care. The details of each record were verified by MBBCh V students, using a defined level-ofcare template and the full discharge summary. The data were reviewed by a senior clinician. There were 3 007 discharge summaries - 97 were not classifiable, two were test records and one was a duplicate. These 100 records were excluded. There were no primary-level records. Secondary-level patients represented 29% (854) of those discharged and 19% of total bed days. Tertiary- and quaternary-level patients together represented 71% of the total and 81% of bed days. The average length of stay was 4.31 days for secondary, 6.98 days for tertiary and 9.77 days for quaternary level-of-care allocation. Almost one-third (29%) of patients discharged from CMJAH's Department of Surgery were deemed suitable for secondarylevel care. These patients had a shorter length of stay and comprised 19% of total bed days. Students and electronic databases represent an important research resource.

In vivo evaluation of axial integrity of coronary stents using intravascular ultrasound: Insights on longitudinal stent deformation.

PubMed

Dvir, Danny; Kitabata, Hironori; Barbash, Israel M; Minha, Sa'ar; Badr, Salem; Loh, Joshua P; Chen, Fang; Torguson, Rebecca; Waksman, Ron

2014-09-01

To evaluate the axial integrity of different coronary stents using intravascular ultrasound (IVUS). Longitudinal stent deformation was recently reported. Consecutive patients who underwent IVUS analysis after drug-eluting stent (DES) implantation for de novo coronary lesions were evaluated. Stent length was compared with label length for calculation of absolute change and relative difference (absolute change divided by label length). A total of 233 DES utilizing five different platforms were included. The median absolute change in stent length was 0.90 mm (interquartile range [IQR] 0.48-1.39) and the relative difference was 5.24% (IQR 2.55-8.29). There was no significant difference among the groups in median absolute or relative change: Cypher 0.89 mm/3.89%, Taxus 0.88 mm/5.39%, Endeavor 1.16 mm/6.77%, Xience V 0.86 mm/5.80%, and PROMUS Element 0.79 mm/5.34% (P = 0.085, P = 0.072, respectively). Multivariate logistic regression revealed that the Cypher stent was independently correlated with a lower change in length, whereas stent label length and deployment pressure were correlated with higher absolute change. The axial integrity of DES platforms examined in vivo was high, with only mild changes in stent length after implantation. While there are differences between first- and second-generation DES, axial integrity among second-generation DES was similar. © 2013 Wiley Periodicals, Inc.

Measurement of the Length of Installed Rock Bolt Based on Stress Wave Reflection by Using a Giant Magnetostrictive (GMS) Actuator and a PZT Sensor.

PubMed

Luo, Mingzhang; Li, Weijie; Wang, Bo; Fu, Qingqing; Song, Gangbing

2017-02-23

Rock bolts, as a type of reinforcing element, are widely adopted in underground excavations and civil engineering structures. Given the importance of rock bolts, the research outlined in this paper attempts to develop a portable non-destructive evaluation method for assessing the length of installed rock bolts for inspection purposes. Traditionally, piezoelectric elements or hammer impacts were used to perform non-destructive evaluation of rock bolts. However, such methods suffered from many major issues, such as the weak energy generated and the requirement for permanent installation for piezoelectric elements, and the inconsistency of wave generation for hammer impact. In this paper, we proposed a portable device for the non-destructive evaluation of rock bolt conditions based on a giant magnetostrictive (GMS) actuator. The GMS actuator generates enough energy to ensure multiple reflections of the stress waves along the rock bolt and a lead zirconate titantate (PZT) sensor is used to detect the reflected waves. A new integrated procedure that involves correlation analysis, wavelet denoising, and Hilbert transform was proposed to process the multiple reflection signals to determine the length of an installed rock bolt. The experimental results from a lab test and field tests showed that, by analyzing the instant phase of the periodic reflections of the stress wave generated by the GMS transducer, the length of an embedded rock bolt can be accurately determined.

Measurement of the Length of Installed Rock Bolt Based on Stress Wave Reflection by Using a Giant Magnetostrictive (GMS) Actuator and a PZT Sensor

PubMed Central

Luo, Mingzhang; Li, Weijie; Wang, Bo; Fu, Qingqing; Song, Gangbing

2017-01-01

Rock bolts, as a type of reinforcing element, are widely adopted in underground excavations and civil engineering structures. Given the importance of rock bolts, the research outlined in this paper attempts to develop a portable non-destructive evaluation method for assessing the length of installed rock bolts for inspection purposes. Traditionally, piezoelectric elements or hammer impacts were used to perform non-destructive evaluation of rock bolts. However, such methods suffered from many major issues, such as the weak energy generated and the requirement for permanent installation for piezoelectric elements, and the inconsistency of wave generation for hammer impact. In this paper, we proposed a portable device for the non-destructive evaluation of rock bolt conditions based on a giant magnetostrictive (GMS) actuator. The GMS actuator generates enough energy to ensure multiple reflections of the stress waves along the rock bolt and a lead zirconate titantate (PZT) sensor is used to detect the reflected waves. A new integrated procedure that involves correlation analysis, wavelet denoising, and Hilbert transform was proposed to process the multiple reflection signals to determine the length of an installed rock bolt. The experimental results from a lab test and field tests showed that, by analyzing the instant phase of the periodic reflections of the stress wave generated by the GMS transducer, the length of an embedded rock bolt can be accurately determined. PMID:28241503

Design, fabrication, and testing of an external fuel (UO2), full-length thermionic converter

NASA Technical Reports Server (NTRS)

Schock, A.; Raab, B.

1971-01-01

The development of a full-length external-fuel thermionic converter for in-pile testing is described. The development program includes out-of-pile performance testing of the fully fueled-converter, using RF-induction heating, before its installation in the in-pile test capsule. The external-fuel converter is cylindrical in shape, and consists of an inner, centrally cooled collector, and an outer emitter surrounded by nuclear fuel. The term full-length denotes that the converter is long enough to extend over the full height of the reactor core. Thus, the converter is not a scaled-down test device, but a full-scale fuel element of the thermionic reactor. The external-fuel converter concept permits a number of different design options, particularly with respect to the fuel composition and shape, and the collector cooling arrangement. The converter described was developed for the Jet Propulsion Laboratory, and is based on their concept for a thermionic reactor with uninsulated collector cooling as previously described. The converter is double-ended, with through-flow cooling, and with ceramic seals and emitter and collector power take-offs at both ends. The design uses a revolver-shaped tungsten emitter body, with the central emitter hole surrounded by six peripheral fuel holes loaded with cylindrical UO2 pellets.

Scaling high-order harmonic generation from laser-solid interactions to ultrahigh intensity.

PubMed

Dollar, F; Cummings, P; Chvykov, V; Willingale, L; Vargas, M; Yanovsky, V; Zulick, C; Maksimchuk, A; Thomas, A G R; Krushelnick, K

2013-04-26

Coherent x-ray beams with a subfemtosecond (<10(-15)  s) pulse duration will enable measurements of fundamental atomic processes in a completely new regime. High-order harmonic generation (HOHG) using short pulse (<100  fs) infrared lasers focused to intensities surpassing 10(18)  W cm(-2) onto a solid density plasma is a promising means of generating such short pulses. Critical to the relativistic oscillating mirror mechanism is the steepness of the plasma density gradient at the reflection point, characterized by a scale length, which can strongly influence the harmonic generation mechanism. It is shown that for intensities in excess of 10(21)  W cm(-2) an optimum density ramp scale length exists that balances an increase in efficiency with a growth of parametric plasma wave instabilities. We show that for these higher intensities the optimal scale length is c/ω0, for which a variety of HOHG properties are optimized, including total conversion efficiency, HOHG divergence, and their power law scaling. Particle-in-cell simulations show striking evidence of the HOHG loss mechanism through parametric instabilities and relativistic self-phase modulation, which affect the produced spectra and conversion efficiency.

A random spatial network model based on elementary postulates

USGS Publications Warehouse

Karlinger, Michael R.; Troutman, Brent M.

1989-01-01

A model for generating random spatial networks that is based on elementary postulates comparable to those of the random topology model is proposed. In contrast to the random topology model, this model ascribes a unique spatial specification to generated drainage networks, a distinguishing property of some network growth models. The simplicity of the postulates creates an opportunity for potential analytic investigations of the probabilistic structure of the drainage networks, while the spatial specification enables analyses of spatially dependent network properties. In the random topology model all drainage networks, conditioned on magnitude (number of first-order streams), are equally likely, whereas in this model all spanning trees of a grid, conditioned on area and drainage density, are equally likely. As a result, link lengths in the generated networks are not independent, as usually assumed in the random topology model. For a preliminary model evaluation, scale-dependent network characteristics, such as geometric diameter and link length properties, and topologic characteristics, such as bifurcation ratio, are computed for sets of drainage networks generated on square and rectangular grids. Statistics of the bifurcation and length ratios fall within the range of values reported for natural drainage networks, but geometric diameters tend to be relatively longer than those for natural networks.

Compressible pulsating convection through regular and random porous media: the thermoacoustic case

NASA Astrophysics Data System (ADS)

Tasnim, Syeda Humaira; Mahmud, Shohel; Fraser, Roydon Andrew

2012-02-01

The effects of material, geometry, length and position of the porous channels on energy transfer in air-filled enclosures carrying a compressible pulsating wave are investigated. The pulsating fluid motion is created by an acoustic driver in a resonant chamber. Three different porous materials (Corning Celcor, Reticulated Vitreous Carbon (RVC), and Mylar plastic), three different geometries (square, open foam, and circular cross-section), six different lengths, " L" (varying between 1 and 6.5 cm, L = 0.01-0.068 λ, where λ is the wavelength of the fundamental acoustic mode), and eight different positions (hot end of the channel, varying between 0.5 and 8 cm) of the channels from the pressure anti-node is experimentally measured. The surface temperature distribution on the channel wall and temperature difference generated across the channel walls are measured while energy flow along the channel walls is calculated analytically. The experimental results are compared with a 1-D numerical code and found excellent agreement. The material, geometry, length, and position of the porous channel strongly affect the energy interactions between the porous channel and the working fluid. The temperature difference generated across the porous RVC channel increases as the porosity increases form 20 to 80 PPI; but decreases if the porosity increases further. Corning Celcor shows improved temperature difference generated across the channel as the length of the channel increases; but then decreases if the length is further increased. The results of this study are applicable to the design of thermoacoustic devices.

Low-threshold collinear parametric Raman comb generation in calcite under 532 and 1064 nm picosecond laser pumping

NASA Astrophysics Data System (ADS)

Smetanin, S. N.; Jelínek, M., Jr.; Kubeček, V.; Jelínková, H.

2015-09-01

Optimal conditions of low-threshold collinear parametric Raman comb generation in calcite (CaCO3) are experimentally investigated under 20 ps laser pulse excitation, in agreement with the theoretical study. The collinear parametric Raman generation of the highest number of Raman components in the short calcite crystals corresponding to the optimal condition of Stokes-anti-Stokes coupling was achieved. At the excitation wavelength of 1064 nm, using the optimum-length crystal resulted in the effective multi-octave frequency Raman comb generation containing up to five anti-Stokes and more than four Stokes components (from 674 nm to 1978 nm). The 532 nm pumping resulted in the frequency Raman comb generation from the 477 nm 2nd anti-Stokes up to the 692 nm 4th Stokes component. Using the crystal with a non-optimal length leads to the Stokes components generation only with higher thresholds because of the cascade-like stimulated Raman scattering with suppressed parametric coupling.

The Effect of Commonly Performed Exercises on the Levator Hiatus Area and the Length and Strength of Pelvic Floor Muscles in Postpartum Women.

PubMed

Siff, Lauren N; Hill, Audra J; Walters, Samantha J; Walters, Ginny; Walters, Mark D

2018-05-02

The aim oft his study was to compare the effects of 10 common exercises to traditional pelvic floor muscle (PFM) contractions (Kegel) on levator hiatus (LH) area and PFM length and strength. This is a cross-sectional study of 15 healthy postpartum women. Ten exercises were studied. These were common variations of leg, core, and back exercises used in yoga, Pilates, strength training, and physical therapy. Each participant performed all 10 exercises at a single visit in 2 examination settings: transperineal ultrasound and perineometry. Ultrasound measured the LH area and PFM length, and perineometry measured the muscle strength (peak squeeze pressure). Kegel generates an increase in squeeze pressure (24.3 cm H2O), shortens the muscles (-0.46 cm) and narrows the LH (-0.13 cm). The bird-dog and plank exercises were not different from Kegel in any measurement. While the leg-lift ultrasound dimensions are similar to Kegel, leg lifts generated peak squeeze pressures stronger than any other exercise (including Kegel). Whereas ultrasound dimensions were similar to Kegel, tucked and untucked squats and thigh adductions generated weaker contractions than Kegel. While crunch generated a squeeze pressure similar to Kegel, the ultrasound dimensions showed a significantly wider LH and longer muscle than Kegel. Bridge, clam, and plié exercises affected the PFMs differently than Kegel in all measures. Bird-dog, plank, and leg-lift exercises should be evaluated as alternative exercises to Kegel as they affect PFM strength and length and LH area similarly to Kegel, and leg lifts generate a stronger contraction than Kegel.

Longer guts and higher food quality increase energy intake in migratory swans.

PubMed

van Gils, Jan A; Beekman, Jan H; Coehoorn, Pieter; Corporaal, Els; Dekkers, Ten; Klaassen, Marcel; van Kraaij, Rik; de Leeuw, Rinze; de Vries, Peter P

2008-11-01

1. Within the broad field of optimal foraging, it is increasingly acknowledged that animals often face digestive constraints rather than constraints on rates of food collection. This therefore calls for a formalization of how animals could optimize food absorption rates. 2. Here we generate predictions from a simple graphical optimal digestion model for foragers that aim to maximize their (true) metabolizable food intake over total time (i.e. including nonforaging bouts) under a digestive constraint. 3. The model predicts that such foragers should maintain a constant food retention time, even if gut length or food quality changes. For phenotypically flexible foragers, which are able to change the size of their digestive machinery, this means that an increase in gut length should go hand in hand with an increase in gross intake rate. It also means that better quality food should be digested more efficiently. 4. These latter two predictions are tested in a large avian long-distance migrant, the Bewick's swan (Cygnus columbianus bewickii), feeding on grasslands in its Dutch wintering quarters. 5. Throughout winter, free-ranging Bewick's swans, growing a longer gut and experiencing improved food quality, increased their gross intake rate (i.e. bite rate) and showed a higher digestive efficiency. These responses were in accordance with the model and suggest maintenance of a constant food retention time. 6. These changes doubled the birds' absorption rate. Had only food quality changed (and not gut length), then absorption rate would have increased by only 67%; absorption rate would have increased by only 17% had only gut length changed (and not food quality). 7. The prediction that gross intake rate should go up with gut length parallels the mechanism included in some proximate models of foraging that feeding motivation scales inversely to gut fullness. We plea for a tighter integration between ultimate and proximate foraging models.

LoRTE: Detecting transposon-induced genomic variants using low coverage PacBio long read sequences.

PubMed

Disdero, Eric; Filée, Jonathan

2017-01-01

Population genomic analysis of transposable elements has greatly benefited from recent advances of sequencing technologies. However, the short size of the reads and the propensity of transposable elements to nest in highly repeated regions of genomes limits the efficiency of bioinformatic tools when Illumina or 454 technologies are used. Fortunately, long read sequencing technologies generating read length that may span the entire length of full transposons are now available. However, existing TE population genomic softwares were not designed to handle long reads and the development of new dedicated tools is needed. LoRTE is the first tool able to use PacBio long read sequences to identify transposon deletions and insertions between a reference genome and genomes of different strains or populations. Tested against simulated and genuine Drosophila melanogaster PacBio datasets, LoRTE appears to be a reliable and broadly applicable tool to study the dynamic and evolutionary impact of transposable elements using low coverage, long read sequences. LoRTE is an efficient and accurate tool to identify structural genomic variants caused by TE insertion or deletion. LoRTE is available for download at http://www.egce.cnrs-gif.fr/?p=6422.

Cloning of TPS gene from eelgrass species Zostera marina and its functional identification by genetic transformation in rice.

PubMed

Zhao, Feng; Li, Qiuying; Weng, Manli; Wang, Xiuliang; Guo, Baotai; Wang, Li; Wang, Wei; Duan, Delin; Wang, Bin

2013-12-01

The full-length cDNA sequence (2613 bp) of the trehalose-6-phosphate synthase (TPS) gene of eelgrass Zostera marina (ZmTPS) was identified and cloned. Z. marina is a kind of seed-plant growing in sea water during its whole life history. The open reading frame (ORF) region of ZmTPS gene encodes a protein of 870 amino acid residues and a stop codon. The corresponding genomic DNA sequence is 3770 bp in length, which contains 3 exons and 2 introns. The ZmTPS gene was transformed into rice variety ZH11 via Agrobacterium-mediated transformation method. After antibiotic screening, molecular characterization, salt-tolerance and trehalose content determinations, two transgenic lines resistant to 150 mM NaCL solutions were screened. Our study results indicated that the ZmTPS gene was integrated into the genomic DNA of the two transgenic rice lines and could be expressed well. Moreover, the detection of the transformed ZmTPS gene in the progenies of the two transgenic lines was performed from T1 to T4 generations; and results suggested that the transformed ZmTPS gene can be transmitted from parent to the progeny in transgenic rice. © 2013.

Advantages of genome sequencing by long-read sequencer using SMRT technology in medical area.

PubMed

Nakano, Kazuma; Shiroma, Akino; Shimoji, Makiko; Tamotsu, Hinako; Ashimine, Noriko; Ohki, Shun; Shinzato, Misuzu; Minami, Maiko; Nakanishi, Tetsuhiro; Teruya, Kuniko; Satou, Kazuhito; Hirano, Takashi

2017-07-01

PacBio RS II is the first commercialized third-generation DNA sequencer able to sequence a single molecule DNA in real-time without amplification. PacBio RS II's sequencing technology is novel and unique, enabling the direct observation of DNA synthesis by DNA polymerase. PacBio RS II confers four major advantages compared to other sequencing technologies: long read lengths, high consensus accuracy, a low degree of bias, and simultaneous capability of epigenetic characterization. These advantages surmount the obstacle of sequencing genomic regions such as high/low G+C, tandem repeat, and interspersed repeat regions. Moreover, PacBio RS II is ideal for whole genome sequencing, targeted sequencing, complex population analysis, RNA sequencing, and epigenetics characterization. With PacBio RS II, we have sequenced and analyzed the genomes of many species, from viruses to humans. Herein, we summarize and review some of our key genome sequencing projects, including full-length viral sequencing, complete bacterial genome and almost-complete plant genome assemblies, and long amplicon sequencing of a disease-associated gene region. We believe that PacBio RS II is not only an effective tool for use in the basic biological sciences but also in the medical/clinical setting.

History-dependence of muscle slack length following contraction and stretch in the human vastus lateralis.

PubMed

Stubbs, Peter W; Walsh, Lee D; D'Souza, Arkiev; Héroux, Martin E; Bolsterlee, Bart; Gandevia, Simon C; Herbert, Robert D

2018-06-01

In reduced muscle preparations, the slack length and passive stiffness of muscle fibres have been shown to be influenced by previous muscle contraction or stretch. In human muscles, such behaviours have been inferred from measures of muscle force, joint stiffness and reflex magnitudes and latencies. Using ultrasound imaging, we directly observed that isometric contraction of the vastus lateralis muscle at short lengths reduces the slack lengths of the muscle-tendon unit and muscle fascicles. The effect is apparent 60 s after the contraction. These observations imply that muscle contraction at short lengths causes the formation of bonds which reduce the effective length of structures that generate passive tension in muscles. In reduced muscle preparations, stretch and muscle contraction change the properties of relaxed muscle fibres. In humans, effects of stretch and contraction on properties of relaxed muscles have been inferred from measurements of time taken to develop force, joint stiffness and reflex latencies. The current study used ultrasound imaging to directly observe the effects of stretch and contraction on muscle-tendon slack length and fascicle slack length of the human vastus lateralis muscle in vivo. The muscle was conditioned by (a) strong isometric contractions at long muscle-tendon lengths, (b) strong isometric contractions at short muscle-tendon lengths, (c) weak isometric contractions at long muscle-tendon lengths and (d) slow stretches. One minute after conditioning, ultrasound images were acquired from the relaxed muscle as it was slowly lengthened through its physiological range. The ultrasound image sequences were used to identify muscle-tendon slack angles and fascicle slack lengths. Contraction at short muscle-tendon lengths caused a mean 13.5 degree (95% CI 11.8-15.0 degree) shift in the muscle-tendon slack angle towards shorter muscle-tendon lengths, and a mean 5 mm (95% CI 2-8 mm) reduction in fascicle slack length, compared to the other conditions. A supplementary experiment showed the effect could be demonstrated if the muscle was conditioned by contraction at short lengths but not if the relaxed muscle was held at short lengths, confirming the role of muscle contraction. These observations imply that muscle contraction at short lengths causes the formation of bonds which reduce the effective length of structures that generate passive tension in muscles. © 2018 The Authors. The Journal of Physiology © 2018 The Physiological Society.

Stochastic Modeling based on Dictionary Approach for the Generation of Daily Precipitation Occurrences

NASA Astrophysics Data System (ADS)

Panu, U. S.; Ng, W.; Rasmussen, P. F.

2009-12-01

The modeling of weather states (i.e., precipitation occurrences) is critical when the historical data are not long enough for the desired analysis. Stochastic models (e.g., Markov Chain and Alternating Renewal Process (ARP)) of the precipitation occurrence processes generally assume the existence of short-term temporal-dependency between the neighboring states while implying the existence of long-term independency (randomness) of states in precipitation records. Existing temporal-dependent models for the generation of precipitation occurrences are restricted either by the fixed-length memory (e.g., the order of a Markov chain model), or by the reining states in segments (e.g., persistency of homogenous states within dry/wet-spell lengths of an ARP). The modeling of variable segment lengths and states could be an arduous task and a flexible modeling approach is required for the preservation of various segmented patterns of precipitation data series. An innovative Dictionary approach has been developed in the field of genome pattern recognition for the identification of frequently occurring genome segments in DNA sequences. The genome segments delineate the biologically meaningful ``words" (i.e., segments with a specific patterns in a series of discrete states) that can be jointly modeled with variable lengths and states. A meaningful “word”, in hydrology, can be referred to a segment of precipitation occurrence comprising of wet or dry states. Such flexibility would provide a unique advantage over the traditional stochastic models for the generation of precipitation occurrences. Three stochastic models, namely, the alternating renewal process using Geometric distribution, the second-order Markov chain model, and the Dictionary approach have been assessed to evaluate their efficacy for the generation of daily precipitation sequences. Comparisons involved three guiding principles namely (i) the ability of models to preserve the short-term temporal-dependency in data through the concepts of autocorrelation, average mutual information, and Hurst exponent, (ii) the ability of models to preserve the persistency within the homogenous dry/wet weather states through analysis of dry/wet-spell lengths between the observed and generated data, and (iii) the ability to assesses the goodness-of-fit of models through the likelihood estimates (i.e., AIC and BIC). Past 30 years of observed daily precipitation records from 10 Canadian meteorological stations were utilized for comparative analyses of the three models. In general, the Markov chain model performed well. The remainders of the models were found to be competitive from one another depending upon the scope and purpose of the comparison. Although the Markov chain model has a certain advantage in the generation of daily precipitation occurrences, the structural flexibility offered by the Dictionary approach in modeling the varied segment lengths of heterogeneous weather states provides a distinct and powerful advantage in the generation of precipitation sequences.

Improving the Performance of Two-Stage Gas Guns By Adding a Diaphragm in the Pump Tube

NASA Technical Reports Server (NTRS)

Bogdanoff, D. W.; Miller, Robert J.

1995-01-01

Herein, we study the technique of improving the gun performance by installing a diaphragm in the pump tube of the gun. A CFD study is carried out for the 0.28 in. gun in the Hypervelocity Free Flight Radiation (HFF RAD) range at the NASA Ames Research Center. The normal, full-length pump tube is studied as well as two pump tubes of reduced length (approximately 75% and approximately 33% of the normal length). Significant improvements in performance are calculated to be gained for the reduced length pump tubes upon the addition of the diaphragm. These improvements are identified as reductions in maximum pressures in the pump tube and at the projectile base of approximately 20%, while maintaining the projectile muzzle velocity or as increases in muzzle velocity of approximately 0.5 km/sec while not increasing the maximum pressures in the gun. Also, it is found that both guns with reduced pump tube length (with diaphragms) could maintain the performance of gun with the full length pump tube without diaphragms, whereas the guns with reduced pump tube lengths without diaphragms could not. A five-shot experimental investigation of the pump tube diaphragm technique is carried out for the gun with a pump tube length of 75% normal. The CFD predictions of increased muzzle velocity are borne out by the experimental data. Modest, but useful muzzle velocity increases (2.5 - 6%) are obtained upon the installation of a diaphragm, compared to a benchmark shot without a diaphragm.

In vivo Length Change Patterns of the Medial and Lateral Collateral Ligaments along the Flexion Path of the Knee

PubMed Central

Hosseini, Ali; Qi, Wei; Tsai, Tsung-Yuan; Liu, Yujie; Rubash, Harry; Li, Guoan

2014-01-01

Purpose The knowledge of the function of the collateral ligaments – i.e., superficial medial collateral ligament (sMCL), deep medial collateral ligament (dMCL) and lateral collateral ligament (LCL) – in the entire range of knee flexion is important for soft tissue balance during total knee arthroplasty. The objective of this study was to investigate the length changes of different portions (anterior, middle and posterior) of the sMCL, dMCL and LCL during in vivo weightbearing flexion from full extension to maximal knee flexion. Methods Using a dual fluoroscopic imaging system eight healthy knees were imaged while performing a lunge from full extension to maximal flexion. The length changes of each portion of the collateral ligaments were measured along the flexion path of the knee. Results All anterior portions of the collateral ligaments were shown to have increasing length with flexion except that of the sMCL which showed a reduction in length at high flexion. The middle portions showed minimal change in lengths except that of the sMCL which showed a consistent reduction in length with flexion. All posterior portions showed reduction in lengths with flexion. Conclusions These data indicated that every portion of the ligaments may play important roles in knee stability at different knee flexion range. The soft tissue releasing during TKA may need to consider the function of the ligament portions along the entire flexion path including maximum flexion. PMID:25239504

Photoconductivity, pH Sensitivity, Noise, and Channel Length Effects in Si Nanowire FET Sensors

NASA Astrophysics Data System (ADS)

Gasparyan, Ferdinand; Zadorozhnyi, Ihor; Khondkaryan, Hrant; Arakelyan, Armen; Vitusevich, Svetlana

2018-03-01

Silicon nanowire (NW) field-effect transistor (FET) sensors of various lengths were fabricated. Transport properties of Si NW FET sensors were investigated involving noise spectroscopy and current-voltage (I-V) characterization. The static I-V dependencies demonstrate the high quality of fabricated silicon FETs without leakage current. Transport and noise properties of NW FET structures were investigated under different light illumination conditions, as well as in sensor configuration in an aqueous solution with different pH values. Furthermore, we studied channel length effects on the photoconductivity, noise, and pH sensitivity. The magnitude of the channel current is approximately inversely proportional to the length of the current channel, and the pH sensitivity increases with the increase of channel length approaching the Nernst limit value of 59.5 mV/pH. We demonstrate that dominant 1/f-noise can be screened by the generation-recombination plateau at certain pH of the solution or external optical excitation. The characteristic frequency of the generation-recombination noise component decreases with increasing of illumination power. Moreover, it is shown that the measured value of the slope of 1/f-noise spectral density dependence on the current channel length is 2.7 which is close to the theoretically predicted value of 3.

Tankyrase 2 Poly(ADP-Ribose) Polymerase Domain-Deleted Mice Exhibit Growth Defects but Have Normal Telomere Length and Capping

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hsiao, Susan J; Poitras, Marc; Cook, Brandoch

Regulation of telomere length maintenance and capping are a critical cell functions in both normal and tumor cells. Tankyrase 2 (Tnks2) is a poly(ADP-ribose) polymerase (PARP) that has been shown to modify itself and TRF1, a telomere-binding protein. We show here by overexpression studies that tankyrase 2, like its closely related homolog tankyrase 1, can function as a positive regulator of telomere length in human cells, dependent on its catalytic PARP activity. To study the role of Tnks2 in vivo, we generated mice with the Tnks2 PARP domain deleted. These mice are viable and fertile but display a growth retardationmore » phenotype. Telomere analysis by quantitative fluorescence in situ hybridization (FISH), flow-FISH, and restriction fragment analysis showed no change in telomere length or telomere capping in these mice. To determine the requirement foTnks2 in long-term maintenance of telomeres, we generated embryonic stem cells with the Tnks2 PARP domain deleted and observed no change, even upon prolonged growth, in telomere length or telomere capping. Together these results suggest that Tnkjs2 has a role in normal growth and development but is not essential for telomere length maintenance or telomere capping in mice.« less

A common polymorphism of the growth hormone receptor is associated with increased responsiveness to growth hormone.

PubMed

Dos Santos, Christine; Essioux, Laurent; Teinturier, Cécile; Tauber, Maïté; Goffin, Vincent; Bougnères, Pierre

2004-07-01

Growth hormone is used to increase height in short children who are not deficient in growth hormone, but its efficacy varies largely across individuals. The genetic factors responsible for this variation are entirely unknown. In two cohorts of short children treated with growth hormone, we found that an isoform of the growth hormone receptor gene that lacks exon 3 (d3-GHR) was associated with 1.7 to 2 times more growth acceleration induced by growth hormone than the full-length isoform (P < 0.0001). In transfection experiments, the transduction of growth hormone signaling through d3-GHR homo- or heterodimers was approximately 30% higher than through full-length GHR homodimers (P < 0.0001). One-half of Europeans are hetero- or homozygous with respect to the allele encoding the d3-GHR isoform, which is dominant over the full-length isoform. These observations suggest that the polymorphism in exon 3 of GHR is important in growth hormone pharmacogenetics.

Full-length genomic characterization and molecular evolution of canine parvovirus in China.

PubMed

Zhou, Ling; Tang, Qinghai; Shi, Lijun; Kong, Miaomiao; Liang, Lin; Mao, Qianqian; Bu, Bin; Yao, Lunguang; Zhao, Kai; Cui, Shangjin; Leal, Élcio

2016-06-01

Canine parvovirus type 2 (CPV-2) can cause acute haemorrhagic enteritis in dogs and myocarditis in puppies. This disease has become one of the most serious infectious diseases of dogs. During 2014 in China, there were many cases of acute infectious diarrhoea in dogs. Some faecal samples were negative for the CPV-2 antigen based on a colloidal gold test strip but were positive based on PCR, and a viral strain was isolated from one such sample. The cytopathic effect on susceptible cells and the results of the immunoperoxidase monolayer assay, PCR, and sequencing indicated that the pathogen was CPV-2. The strain was named CPV-NY-14, and the full-length genome was sequenced and analysed. A maximum likelihood tree was constructed using the full-length genome and all available CPV-2 genomes. New strains have replaced the original strain in Taiwan and Italy, although the CPV-2a strain is still predominant there. However, CPV-2a still causes many cases of acute infectious diarrhoea in dogs in China.

High-Resolution Sequence-Function Mapping of Full-Length Proteins

PubMed Central

Kowalsky, Caitlin A.; Klesmith, Justin R.; Stapleton, James A.; Kelly, Vince; Reichkitzer, Nolan; Whitehead, Timothy A.

2015-01-01

Comprehensive sequence-function mapping involves detailing the fitness contribution of every possible single mutation to a gene by comparing the abundance of each library variant before and after selection for the phenotype of interest. Deep sequencing of library DNA allows frequency reconstruction for tens of thousands of variants in a single experiment, yet short read lengths of current sequencers makes it challenging to probe genes encoding full-length proteins. Here we extend the scope of sequence-function maps to entire protein sequences with a modular, universal sequence tiling method. We demonstrate the approach with both growth-based selections and FACS screening, offer parameters and best practices that simplify design of experiments, and present analytical solutions to normalize data across independent selections. Using this protocol, sequence-function maps covering full sequences can be obtained in four to six weeks. Best practices introduced in this manuscript are fully compatible with, and complementary to, other recently published sequence-function mapping protocols. PMID:25790064

Structure of an E. coli integral membrane sulfurtransferase and its structural transition upon SCN- binding defined by EPR-based hybrid method

NASA Astrophysics Data System (ADS)

Ling, Shenglong; Wang, Wei; Yu, Lu; Peng, Junhui; Cai, Xiaoying; Xiong, Ying; Hayati, Zahra; Zhang, Longhua; Zhang, Zhiyong; Song, Likai; Tian, Changlin

2016-01-01

Electron paramagnetic resonance (EPR)-based hybrid experimental and computational approaches were applied to determine the structure of a full-length E. coli integral membrane sulfurtransferase, dimeric YgaP, and its structural and dynamic changes upon ligand binding. The solution NMR structures of the YgaP transmembrane domain (TMD) and cytosolic catalytic rhodanese domain were reported recently, but the tertiary fold of full-length YgaP was not yet available. Here, systematic site-specific EPR analysis defined a helix-loop-helix secondary structure of the YagP-TMD monomers using mobility, accessibility and membrane immersion measurements. The tertiary folds of dimeric YgaP-TMD and full-length YgaP in detergent micelles were determined through inter- and intra-monomer distance mapping and rigid-body computation. Further EPR analysis demonstrated the tight packing of the two YgaP second transmembrane helices upon binding of the catalytic product SCN-, which provides insight into the thiocyanate exportation mechanism of YgaP in the E. coli membrane.

Adrenodoxin supports reactions catalyzed by microsomal steroidogenic cytochrome P450s

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pechurskaya, Tatiana A.; Harnastai, Ivan N.; Grabovec, Irina P.

2007-02-16

The interaction of adrenodoxin (Adx) and NADPH cytochrome P450 reductase (CPR) with human microsomal steroidogenic cytochrome P450s was studied. It is found that Adx, mitochondrial electron transfer protein, is able to support reactions catalyzed by human microsomal P450s: full length CYP17, truncated CYP17, and truncated CYP21. CPR, but not Adx, supports activity of truncated CYP19. Truncated and the full length CYP17s show distinct preference for electron donor proteins. Truncated CYP17 has higher activity with Adx compared to CPR. The alteration in preference to electron donor does not change product profile for truncated enzymes. The electrostatic contacts play a major rolemore » in the interaction of truncated CYP17 with either CPR or Adx. Similarly electrostatic contacts are predominant in the interaction of full length CYP17 with Adx. We speculate that Adx might serve as an alternative electron donor for CYP17 at the conditions of CPR deficiency in human.« less

Expression and purification of functional PDGF receptor beta.

PubMed

Shang, Qingbin; Zhao, Liang; Wang, Xiaojing; Wang, Meimei; Sui, Sen-Fang; Mi, Li-Zhi

2017-07-29

Platelet Derived Growth Factor receptors (PDGFRs), members of receptor tyrosine kinase superfamily, play essential roles in early hematopoiesis, angiogenesis and organ development. Dysregulation of PDGF receptor signaling under pathological conditions associates with cancers, vascular diseases, and fibrotic diseases. Therefore, they are attractive targets in drug development. Like any other membrane proteins with a single-pass transmembrane domain, the high-resolution structural information of the full-length PDGF receptors is still not resolved. It is caused, at least in part, by the technical challenges in the expression and purification of the functional, full-length PDGF receptors. Herein, we reported our experimental details in expression and purification of the full-length PDGFRβ from mammalian cells. We found that purified PDGFRβ remained in two different oligomeric states, presumably the monomer and the dimer, with basal kinase activity in detergent micelles. Addition of PDGF-B promoted dimerization and elevated kinase activity of the receptor, suggesting that purified receptors were functional. Copyright © 2017 Elsevier Inc. All rights reserved.

Production of a full-length infectious GFP-tagged cDNA clone of Beet mild yellowing virus for the study of plant-polerovirus interactions.

PubMed

Stevens, Mark; Viganó, Felicita

2007-04-01

The full-length cDNA of Beet mild yellowing virus (Broom's Barn isolate) was sequenced and cloned into the vector pLitmus 29 (pBMYV-BBfl). The sequence of BMYV-BBfl (5721 bases) shared 96% and 98% nucleotide identity with the other complete sequences of BMYV (BMYV-2ITB, France and BMYV-IPP, Germany respectively). Full-length capped RNA transcripts of pBMYV-BBfl were synthesised and found to be biologically active in Arabidopsis thaliana protoplasts following electroporation or PEG inoculation when the protoplasts were subsequently analysed using serological and molecular methods. The BMYV sequence was modified by inserting DNA that encoded the jellyfish green fluorescent protein (GFP) into the P5 gene close to its 3' end. A. thaliana protoplasts electroporated with these RNA transcripts were biologically active and up to 2% of transfected protoplasts showed GFP-specific fluorescence. The exploitation of these cDNA clones for the study of the biology of beet poleroviruses is discussed.

Crystallization and preliminary X-ray crystallographic analysis of carboxyl-terminal region 4 of SigR from Streptomyces coelicolor A3(2)

PubMed Central

Kim, Keon Young; Kim, Sunmin; Park, Jeong Kuk; Song, HyoJin; Park, SangYoun

2014-01-01

Full-length SigR from Streptomyces coelicolor A3(2) was overexpressed in Escherichia coli, purified and submitted to crystallization trials using either polyethylene glycol 3350 or 4000 as a precipitant. X-ray diffraction data were collected to 2.60 Å resolution under cryoconditions using synchrotron X-rays. The crystal packs in space group P43212, with unit-cell parameters a = b = 42.14, c = 102.02 Å. According to the Matthews coefficient, the crystal asymmetric unit cannot contain the full-length protein. Molecular replacement with the known structures of region 2 and region 4 as independent search models indicates that the crystal contains only the −35 element-binding carboxyl-terminal region 4 of full-length SigR. Mass-spectrometric analysis of the harvested crystal confirms this, suggesting a crystal volume per protein weight (V M) of 2.24 Å3 Da−1 and 45.1% solvent content. PMID:24915084

Structure of the full-length glucagon class B G protein-coupled receptor

PubMed Central

Zhang, Haonan; Qiao, Anna; Yang, Dehua; Yang, Linlin; Dai, Antao; de Graaf, Chris; Reedtz-Runge, Steffen; Dharmarajan, Venkatasubramanian; Zhang, Hui; Han, Gye Won; Grant, Thomas D.; Sierra, Raymond G.; Weierstall, Uwe; Nelson, Garrett; Liu, Wei; Wu, Yanhong; Ma, Limin; Cai, Xiaoqing; Lin, Guangyao; Wu, Xiaoai; Geng, Zhi; Dong, Yuhui; Song, Gaojie; Griffin, Patrick R.; Lau, Jesper; Cherezov, Vadim; Yang, Huaiyu; Hanson, Michael A.; Stevens, Raymond C.; Zhao, Qiang; Jiang, Hualiang; Wang, Ming-Wei; Wu, Beili

2017-01-01

The human glucagon receptor (GCGR) belongs to the class B G protein-coupled receptor (GPCR) family and plays a key role in glucose homeostasis and the pathophysiology of type 2 diabetes. Here we report the 3.0 Å crystal structure of full-length GCGR containing both extracellular domain (ECD) and transmembrane domain (TMD) in an inactive conformation. The two domains are connected by a 12-residue segment termed the ‘stalk’, which adopts a β-strand conformation, instead of forming an α-helix as observed in the previously solved structure of GCGR-TMD. The first extracellular loop (ECL1) exhibits a β-hairpin conformation and interacts with the stalk to form a compact β-sheet structure. Hydrogen/deuterium exchange, disulfide cross-linking and molecular dynamics studies suggest that the stalk and ECL1 play critical roles in modulating peptide ligand binding and receptor activation. These insights into the full-length GCGR structure deepen our understanding about the signaling mechanisms of class B GPCRs. PMID:28514451

Conformational states of the full-length glucagon receptor

PubMed Central

Yang, Linlin; Yang, Dehua; de Graaf, Chris; Moeller, Arne; West, Graham M.; Dharmarajan, Venkatasubramanian; Wang, Chong; Siu, Fai Y.; Song, Gaojie; Reedtz-Runge, Steffen; Pascal, Bruce D.; Wu, Beili; Potter, Clinton S.; Zhou, Hu; Griffin, Patrick R.; Carragher, Bridget; Yang, Huaiyu; Wang, Ming-Wei; Stevens, Raymond C.; Jiang, Hualiang

2015-01-01

Class B G protein-coupled receptors are composed of an extracellular domain (ECD) and a seven-transmembrane (7TM) domain, and their signalling is regulated by peptide hormones. Using a hybrid structural biology approach together with the ECD and 7TM domain crystal structures of the glucagon receptor (GCGR), we examine the relationship between full-length receptor conformation and peptide ligand binding. Molecular dynamics (MD) and disulfide crosslinking studies suggest that apo-GCGR can adopt both an open and closed conformation associated with extensive contacts between the ECD and 7TM domain. The electron microscopy (EM) map of the full-length GCGR shows how a monoclonal antibody stabilizes the ECD and 7TM domain in an elongated conformation. Hydrogen/deuterium exchange (HDX) studies and MD simulations indicate that an open conformation is also stabilized by peptide ligand binding. The combined studies reveal the open/closed states of GCGR and suggest that glucagon binds to GCGR by a conformational selection mechanism. PMID:26227798

The word-length effect in acquired alexia, and real and virtual hemianopia.

PubMed

Sheldon, Claire A; Abegg, Mathias; Sekunova, Alla; Barton, Jason J S

2012-04-01

A word-length effect is often described in pure alexia, with reading time proportional to the number of letters in a word. Given the frequent association of right hemianopia with pure alexia, it is uncertain whether and how much of the word-length effect may be attributable to the hemifield loss. To isolate the contribution of the visual field defect, we simulated hemianopia in healthy subjects with a gaze-contingent paradigm during an eye-tracking experiment. We found a minimal word-length effect of 14 ms/letter for full-field viewing, which increased to 38 ms/letter in right hemianopia and to 31 ms/letter in left hemianopia. We found a correlation between mean reading time and the slope of the word-length effect in hemianopic conditions. The 95% upper prediction limits for the word-length effect were 51 ms/letter in subjects with full visual fields and 161 ms/letter with simulated right hemianopia. These limits, which can be considered diagnostic criteria for an alexic word-length effect, were consistent with the reading performance of six patients with diagnoses based independently on perimetric and imaging data: two patients with probable hemianopic dyslexia, and four with alexia and lesions of the left fusiform gyrus, two with and two without hemianopia. Two of these patients also showed reduction of the word-length effect over months, one with and one without a reading rehabilitation program. Our findings clarify the magnitude of the word-length effect that originates from hemianopia alone, and show that the criteria for a word-length effect indicative of alexia differ according to the degree of associated hemifield loss. Copyright © 2012 Elsevier Ltd. All rights reserved.

A DN-mda5 transgenic zebrafish model demonstrates that Mda5 plays an important role in snakehead rhabdovirus resistance.

PubMed

Gabor, K A; Charette, J R; Pietraszewski, M J; Wingfield, D J; Shim, J S; Millard, P J; Kim, C H

2015-08-01

Melanoma Differentiation-Associated protein 5 (MDA5) is a member of the retinoic acid-inducible gene I (RIG-I)-like receptor (RLR) family, which is a cytosolic pattern recognition receptor that detects viral nucleic acids. Here we show an Mda5-dependent response to rhabdovirus infection in vivo using a dominant-negative mda5 transgenic zebrafish. Dominant-negative mda5 zebrafish embryos displayed an impaired antiviral immune response compared to wild-type counterparts that can be rescued by recombinant full-length Mda5. To our knowledge, we have generated the first dominant-negative mda5 transgenic zebrafish and demonstrated a critical role for Mda5 in the antiviral response to rhabdovirus. Copyright © 2015 Elsevier Ltd. All rights reserved.

What can we learn about lyssavirus genomes using 454 sequencing?

PubMed

Höper, Dirk; Finke, Stefan; Freuling, Conrad M; Hoffmann, Bernd; Beer, Martin

2012-01-01

The main task of the individual project number four"Whole genome sequencing, virus-host adaptation, and molecular epidemiological analyses of lyssaviruses "within the network" Lyssaviruses--a potential re-emerging public health threat" is to provide high quality complete genome sequences from lyssaviruses. These sequences are analysed in-depth with regard to the diversity of the viral populations as to both quasi-species and so-called defective interfering RNAs. Moreover, the sequence data will facilitate further epidemiological analyses, will provide insight into the evolution of lyssaviruses and will be the basis for the design of novel nucleic acid based diagnostics. The first results presented here indicate that not only high quality full-length lyssavirus genome sequences can be generated, but indeed efficient analysis of the viral population gets feasible.

Synthesis of Phosphatidylserine and Its Stereoisomers: Their Role in Activation of Blood Coagulation.

PubMed

Mallik, Suman; Prasad, Ramesh; Bhattacharya, Anindita; Sen, Prosenjit

2018-05-10

Natural phosphatidylserine (PS), which contains two chiral centers, enhances blood coagulation. However, the process by which PS enhanced blood coagulation is not completely understood. An efficient and flexible synthetic route has been developed to synthesize all of the possible stereoisomers of PS. In this study, we examined the role of PS chiral centers in modulating the activity of the tissue factor (TF)-factor VIIa coagulation initiation complex. Full length TF was relipidated with phosphatidylcholine, and the synthesized PS isomers were individually used to estimate the procoagulant activity of the TF-FVIIa complex via a FXa generation assay. The results revealed that the initiation complex activity was stereoselective and had increased sensitivity to the configuration of the PS glycerol backbone due to optimal protein-lipid interactions.

Design of a nanopatterned long focal-length planar focusing collector for concentrated solar power

NASA Astrophysics Data System (ADS)

Ding, Qing; Choubal, Aakash; Toussaint, Kimani C.

2017-02-01

Concentrated solar power (CSP) facilities heavily utilize parabolic troughs to collect and concentrate sunlight onto receivers that deliver solar thermal energy to heat engines for generating electricity. However, parabolic troughs are bulky and heavy and result in a large capital investment for CSP plants, thereby making it difficult for CSP technology to be competitive with photovoltaics. We present the design of a planar focusing collector (PFC) with focal length beyond the micron scale. The PFC design is based on the use of a nanostructured silver surface for linearly polarized singlewavelength light. The designed PFC consists of metallic nanogrooves on a dielectric substrate. The geometric properties, namely the width and depth, of a single-unit nanogroove allows for full control of the optical phase at desired spatial coordinates along the nanogroove short-axis for a single wavelength. Moreover, we show numerically that such phase control can be used to construct a phase front that mimics that of a cylindrical lens. In addition, we determine the concentration ratio by comparing the width of our PFC design to the cross-sectional width of its focal spot. We also determine the conversion efficiency at long focal lengths by evaluating the ratio of the collected optical power to the incoming optical power. Finally, we examine the focusing behavior across multiple wavelengths and angles of incidence. Our work shows how nano-optics and plasmonics could contribute to this important area of CSP technology.

Effect of a prehop on the muscle-tendon interaction during vertical jumps.

PubMed

Aeles, Jeroen; Lichtwark, Glen; Peeters, Dries; Delecluse, Christophe; Jonkers, Ilse; Vanwanseele, Benedicte

2018-05-01

Many movements use stretch-shortening cycles of a muscle-tendon unit (MTU) for storing and releasing elastic energy. The required stretching of medial gastrocnemius (MG) tendinous tissue during jumps, however, requires large length changes of the muscle fascicles because of the lack of MTU length changes. This has a negative impact on the force-generating capacity of the muscle fascicles. The purpose of this study was to induce a MG MTU stretch before shortening by adding a prehop to the squat jump. Eleven well-trained athletes specialized in jumping performed a prehop squat jump (PHSJ) and a standard squat jump (SSJ). Kinematic data were collected using a 3D motion capture system and were used in a musculoskeletal model to calculate MTU lengths. B-mode ultrasonography of the MG was used to measure fascicle length and pennation angle during the jumps. By combining the muscle-tendon unit lengths, fascicle lengths, and pennation angles, the stretch and recoil of the series elastic element of MG were calculated using a simple geometric muscle-tendon model. Our results show less length changes of the muscle fascicles during the upward motion and lower maximal shortening velocities, increasing the moment-generating capacity of the plantar flexors, reflected in the higher ankle joint moment in the PHSJ compared with the SSJ. Although muscle-tendon interaction during the PHSJ was more optimal, athletes were not able to increase their jump height compared with the SSJ. NEW & NOTEWORTHY This is the first study that aimed to improve the muscle-tendon interaction in squat jumping. We effectively introduced a stretch to the medial gastrocnemius muscle-tendon unit resulting in lower maximal shortening velocities and thus an increase in the plantar flexor force-generating capacity, reflected in the higher ankle joint moment in the prehop squat jump compared with the standard squat jump. Here, we demonstrate an effective method for mechanical optimization of the muscle-tendon interaction in the medial gastrocnemius during squat jumping.

Database-independent Protein Sequencing (DiPS) Enables Full-length de Novo Protein and Antibody Sequence Determination.

PubMed

Savidor, Alon; Barzilay, Rotem; Elinger, Dalia; Yarden, Yosef; Lindzen, Moshit; Gabashvili, Alexandra; Adiv Tal, Ophir; Levin, Yishai

2017-06-01

Traditional "bottom-up" proteomic approaches use proteolytic digestion, LC-MS/MS, and database searching to elucidate peptide identities and their parent proteins. Protein sequences absent from the database cannot be identified, and even if present in the database, complete sequence coverage is rarely achieved even for the most abundant proteins in the sample. Thus, sequencing of unknown proteins such as antibodies or constituents of metaproteomes remains a challenging problem. To date, there is no available method for full-length protein sequencing, independent of a reference database, in high throughput. Here, we present Database-independent Protein Sequencing, a method for unambiguous, rapid, database-independent, full-length protein sequencing. The method is a novel combination of non-enzymatic, semi-random cleavage of the protein, LC-MS/MS analysis, peptide de novo sequencing, extraction of peptide tags, and their assembly into a consensus sequence using an algorithm named "Peptide Tag Assembler." As proof-of-concept, the method was applied to samples of three known proteins representing three size classes and to a previously un-sequenced, clinically relevant monoclonal antibody. Excluding leucine/isoleucine and glutamic acid/deamidated glutamine ambiguities, end-to-end full-length de novo sequencing was achieved with 99-100% accuracy for all benchmarking proteins and the antibody light chain. Accuracy of the sequenced antibody heavy chain, including the entire variable region, was also 100%, but there was a 23-residue gap in the constant region sequence. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Femtosecond X-ray Diffraction: Applications for Laser-Irradiated Materials

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wark, Justin S.

2009-09-10

Over the past few years short pulse x-ray diffraction at the nanosecond and picosecond level has become an established technique in many high-power laser laboratories for interrogating the lattice response of laser-perturbed and shocked matter, and is now finding applications in diagnosing the state of crystalline materials subject to quasi-isentropic compression. We review some of the previous results obtained in this area, for example the direct observation of coherent phonons, the first direct confirmation of the alpha-epsilon transition in shocked iron, and recent measurements indicating that the strength of matter can be measured at shock pressures exceeding a Mbar. Themore » majority of sources used to date have been laser-plasma based, with some work being performed using 3{sup rd} generation synchrotron sources. However, the development of 4{sup th} generation x-ray free-electron lasers, such as LCLS, afford many new opportunities, with pulse lengths in the femtosecond regime. The extremely low divergence and monochromatic nature of the LCLS beam make it well suited to study compressed polycrystalline matter, especially samples with small grain sizes. At extremely short pulse lengths, such that the pulse is shorter than an x-ray extinction depth traversal time, the diffraction process itself becomes time-dependent, and in certain cases the full wave-field solution will be required, particularly if the matter itself is being rapidly perturbed, as will occur if the intense x-ray radiation is used to create warm dense matter, as in recent experiments on FLASH at DESY.« less

Electrochemical cell apparatus having axially distributed entry of a fuel-spent fuel mixture transverse to the cell lengths

DOEpatents

Reichner, Philip; Dollard, Walter J.

1991-01-01

An electrochemical apparatus (10) is made having a generator section (22) containing axially elongated electrochemical cells (16), a fresh gaseous feed fuel inlet (28), a gaseous feed oxidant inlet (30), and at least one gaseous spent fuel exit channel (46), where the spent fuel exit channel (46) passes from the generator chamber (22) to combine with the fresh feed fuel inlet (28) at a mixing apparatus (50), reformable fuel mixture channel (52) passes through the length of the generator chamber (22) and connects with the mixing apparatus (50), that channel containing entry ports (54) within the generator chamber (22), where the axis of the ports is transverse to the fuel electrode surfaces (18), where a catalytic reforming material is distributed near the reformable fuel mixture entry ports (54).

Entangled photon pair generation by spontaneous parametric down-conversion in finite-length one-dimensional photonic crystals

DOE Office of Scientific and Technical Information (OSTI.GOV)

Centini, M.; Sciscione, L.; Sibilia, C.

A description of spontaneous parametric down-conversion in finite-length one-dimensional nonlinear photonic crystals is developed using semiclassical and quantum approaches. It is shown that if a suitable averaging is added to the semiclassical model, its results are in very good agreement with the quantum approach. We propose two structures made with GaN/AlN that generate both degenerate and nondegenerate entangled photon pairs. Both structures are designed so as to achieve a high efficiency of the nonlinear process.

Plasmid DNA initiates replication of yellow fever vaccine in vitro and elicits virus-specific immune response in mice

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tretyakova, Irina; Nickols, Brian; Hidajat, Rachmat

Yellow fever (YF) causes an acute hemorrhagic fever disease in tropical Africa and Latin America. To develop a novel experimental YF vaccine, we applied iDNA infectious clone technology. The iDNA represents plasmid that encodes the full-length RNA genome of 17D vaccine downstream from a cytomegalovirus (CMV) promoter. The vaccine was designed to transcribe the full-length viral RNA and to launch 17D vaccine virus in vitro and in vivo. Transfection with 10 ng of iDNA plasmid was sufficient to start replication of vaccine virus in vitro. Safety of the parental 17D and iDNA-derived 17D viruses was confirmed in AG129 mice deficientmore » in receptors for IFN-α/β/γ. Finally, direct vaccination of BALB/c mice with a single 20 μg dose of iDNA plasmid resulted in seroconversion and elicitation of virus-specific neutralizing antibodies in animals. We conclude that iDNA immunization approach combines characteristics of DNA and attenuated vaccines and represents a promising vaccination strategy for YF. - Highlights: • The iDNA{sup ®} platform combines advantages of DNA and live attenuated vaccines. • Yellow fever (YF) 17D vaccine was launched from iDNA plasmid in vitro and in vivo. • Safety of iDNA-generated 17D virus was confirmed in AG129 mice. • BALB/c mice seroconverted after a single-dose vaccination with iDNA. • YF virus-neutralizing response was elicited in iDNA-vaccinated mice.« less

Lysine residues K66, K109, and K110 in the bovine foamy virus transactivator protein are required for transactivation and viral replication.

PubMed

Zhang, Suzhen; Cui, Xiaoxu; Li, Jing; Liang, Zhibin; Qiao, Wentao; Tan, Juan

2016-04-01

Bovine foamy virus (BFV) is a complex retrovirus that infects cattle. Like all retroviruses, BFV encodes a transactivator Tas protein (BTas) that increases gene transcription from viral promoters. BFV encodes two promoters that can interact with BTas, a conserved promoter in the 5' long terminal repeat (LTR) and a unique internal promoter (IP). Our previous study showed that BTas is acetylated by p300 at residues K66, K109, and K110, which markedly enhanced the ability of BTas to bind to DNA. However, whether these residues are important for BFV replication was not determined. Therefore, in this study we provide direct evidence that BTas is required for BFV replication and demonstrate that residues K66, K109, and K110 are critical for BTas function and BFV replication. Full-length infectious clones were generated, which were BTas deficient or contained lysine to arginine (K→R) mutations at position 66, 109, and/or 110. In vivo data indicated that K→R mutations at positions 66, 109, and 110 in BTas impaired transactivation of both the LTR and IP promoters. In addition, the K→R mutations in full-length infectious clones reduced expression of viral proteins, and the triple mutant and BTas deletion completely abrogated viral replication. Taken together, these results indicate that lysine residues at positions 66, 109, and 110 in the BTas protein are crucial for BFV replication and suggest a potential role for BTas acetylation in regulating the viral life cycle.

Stable expression of calpain 3 from a muscle transgene in vivo: Immature muscle in transgenic mice suggests a role for calpain 3 in muscle maturation

PubMed Central

Spencer, M. J.; Guyon, J. R.; Sorimachi, H.; Potts, A.; Richard, I.; Herasse, M.; Chamberlain, J.; Dalkilic, I.; Kunkel, L. M.; Beckmann, J. S.

2002-01-01

Limb-girdle muscular dystrophy, type 2A (LGMD 2A), is an autosomal recessive disorder that causes late-onset muscle-wasting, and is due to mutations in the muscle-specific protease calpain 3 (C3). Although LGMD 2A would be a feasible candidate for gene therapy, the reported instability of C3 in vitro raised questions about the potential of obtaining a stable, high-level expression of C3 from a transgene in vivo. We have generated transgenic (Tg) mice with muscle-specific overexpression of full-length C3 or C3 isoforms, which arise from alternative splicing, to test whether stable expression of C3 transgenes could occur in vivo. Unexpectedly, we found that full-length C3 can be overexpressed at high levels in vivo, without toxicity. In addition, we found that Tg expressing C3 lacking exon 6, an isoform expressed embryonically, have muscles that resemble regenerating or developing muscle. Tg expressing C3 lacking exon 15 shared this morphology in the soleus, but not other muscles. Assays of inflammation or muscle membrane damage indicated that the Tg muscles were not degenerative, suggesting that the immature muscle resulted from a developmental block rather than degeneration and regeneration. These studies show that C3 can be expressed stably in vivo from a transgene, and indicate that alternatively spliced C3 isoforms should not be used in gene-therapy applications because they impair proper muscle development. PMID:12084932

Construction of a full-length cDNA Library from Chinese oak silkworm pupa and identification of a KK-42-binding protein gene in relation to pupa-diapause termination.

PubMed

Li, Yu-Ping; Xia, Run-Xi; Wang, Huan; Li, Xi-Sheng; Liu, Yan-Qun; Wei, Zhao-Jun; Lu, Cheng; Xiang, Zhong-Huai

2009-06-24

In this study we successfully constructed a full-length cDNA library from Chinese oak silkworm, Antheraea pernyi, the most well-known wild silkworm used for silk production and insect food. Total RNA was extracted from a single fresh female pupa at the diapause stage. The titer of the library was 5 x 10(5) cfu/ml and the proportion of recombinant clones was approximately 95%. Expressed sequence tag (EST) analysis was used to characterize the library. A total of 175 clustered ESTs consisting of 24 contigs and 151 singlets were generated from 250 effective sequences. Of the 175 unigenes, 97 (55.4%) were known genes but only five from A. pernyi, 37 (21.2%) were known ESTs without function annotation, and 41 (23.4%) were novel ESTs. By EST sequencing, a gene coding KK-42-binding protein in A. pernyi (named as ApKK42-BP; GenBank accession no. FJ744151) was identified and characterized. Protein sequence analysis showed that ApKK42-BP was not a membrane protein but an extracellular protein with a signal peptide at position 1-18, and contained two putative conserved domains, abhydro_lipase and abhydrolase_1, suggesting it may be a member of lipase superfamily. Expression analysis based on number of ESTs showed that ApKK42-BP was an abundant gene in the period of diapause stage, suggesting it may also be involved in pupa-diapause termination.

Construction of a full-length cDNA Library from Chinese oak silkworm pupa and identification of a KK-42-binding protein gene in relation to pupa-diapause termination

PubMed Central

Li, Yu-Ping; Xia, Run-Xi; Wang, Huan; Li, Xi-Sheng; Liu, Yan-Qun; Wei, Zhao-Jun; Lu, Cheng; Xiang, Zhong-Huai

2009-01-01

In this study we successfully constructed a full-length cDNA library from Chinese oak silkworm, Antheraea pernyi, the most well-known wild silkworm used for silk production and insect food. Total RNA was extracted from a single fresh female pupa at the diapause stage. The titer of the library was 5 × 105 cfu/ml and the proportion of recombinant clones was approximately 95%. Expressed sequence tag (EST) analysis was used to characterize the library. A total of 175 clustered ESTs consisting of 24 contigs and 151 singlets were generated from 250 effective sequences. Of the 175 unigenes, 97 (55.4%) were known genes but only five from A. pernyi, 37 (21.2%) were known ESTs without function annotation, and 41 (23.4%) were novel ESTs. By EST sequencing, a gene coding KK-42-binding protein in A. pernyi (named as ApKK42-BP; GenBank accession no. FJ744151) was identified and characterized. Protein sequence analysis showed that ApKK42-BP was not a membrane protein but an extracellular protein with a signal peptide at position 1-18, and contained two putative conserved domains, abhydro_lipase and abhydrolase_1, suggesting it may be a member of lipase superfamily. Expression analysis based on number of ESTs showed that ApKK42-BP was an abundant gene in the period of diapause stage, suggesting it may also be involved in pupa-diapause termination. PMID:19564928

Molecular characterization of the rhesus rhadinovirus (RRV) ORF4 gene and the RRV complement control protein it encodes.

PubMed

Mark, Linda; Spiller, O Brad; Okroj, Marcin; Chanas, Simon; Aitken, Jim A; Wong, Scott W; Damania, Blossom; Blom, Anna M; Blackbourn, David J

2007-04-01

The diversity of viral strategies to modulate complement activation indicates that this component of the immune system has significant antiviral potential. One example is the Kaposi's sarcoma-associated herpesvirus (KSHV) complement control protein (KCP), which inhibits progression of the complement cascade. Rhesus rhadinovirus (RRV), like KSHV, is a member of the subfamily Gammaherpesvirinae and currently provides the only in vivo model of KSHV pathobiology in primates. In the present study, we characterized the KCP homologue encoded by RRV, RRV complement control protein (RCP). Two strains of RRV have been sequenced to date (H26-95 and 17577), and the RCPs they encode differ substantially in structure: RCP from strain H26-95 has four complement control protein (CCP) domains, whereas RCP from strain 17577 has eight CCP domains. Transcriptional analyses of the RCP gene (ORF4, referred to herein as RCP) in infected rhesus macaque fibroblasts mapped the ends of the transcripts of both strains. They revealed that H26-95 encodes a full-length, unspliced RCP transcript, while 17577 RCP generates a full-length unspliced mRNA and two alternatively spliced transcripts. Western blotting confirmed that infected cells express RCP, and immune electron microscopy disclosed this protein on the surface of RRV virions. Functional studies of RCP encoded by both RRV strains revealed their ability to suppress complement activation by the classical (antibody-mediated) pathway. These data provide the foundation for studies into the biological significance of gammaherpesvirus complement regulatory proteins in a tractable, non-human primate model.

Molecular Characterization of the Rhesus Rhadinovirus (RRV) ORF4 Gene and the RRV Complement Control Protein It Encodes▿

PubMed Central

Mark, Linda; Spiller, O. Brad; Okroj, Marcin; Chanas, Simon; Aitken, Jim A.; Wong, Scott W.; Damania, Blossom; Blom, Anna M.; Blackbourn, David J.

2007-01-01

The diversity of viral strategies to modulate complement activation indicates that this component of the immune system has significant antiviral potential. One example is the Kaposi's sarcoma-associated herpesvirus (KSHV) complement control protein (KCP), which inhibits progression of the complement cascade. Rhesus rhadinovirus (RRV), like KSHV, is a member of the subfamily Gammaherpesvirinae and currently provides the only in vivo model of KSHV pathobiology in primates. In the present study, we characterized the KCP homologue encoded by RRV, RRV complement control protein (RCP). Two strains of RRV have been sequenced to date (H26-95 and 17577), and the RCPs they encode differ substantially in structure: RCP from strain H26-95 has four complement control protein (CCP) domains, whereas RCP from strain 17577 has eight CCP domains. Transcriptional analyses of the RCP gene (ORF4, referred to herein as RCP) in infected rhesus macaque fibroblasts mapped the ends of the transcripts of both strains. They revealed that H26-95 encodes a full-length, unspliced RCP transcript, while 17577 RCP generates a full-length unspliced mRNA and two alternatively spliced transcripts. Western blotting confirmed that infected cells express RCP, and immune electron microscopy disclosed this protein on the surface of RRV virions. Functional studies of RCP encoded by both RRV strains revealed their ability to suppress complement activation by the classical (antibody-mediated) pathway. These data provide the foundation for studies into the biological significance of gammaherpesvirus complement regulatory proteins in a tractable, non-human primate model. PMID:17287274

Triptolide Inhibits the AR Signaling Pathway to Suppress the Proliferation of Enzalutamide Resistant Prostate Cancer Cells.

PubMed

Han, Yangyang; Huang, Weiwei; Liu, Jiakuan; Liu, Dandan; Cui, Yangyan; Huang, Ruimin; Yan, Jun; Lei, Ming

2017-01-01

Enzalutamide is a second-generation androgen receptor (AR) antagonist for the treatment of metastatic castration-resistant prostate cancer (mCRPC). Unfortunately, AR dysfunction means that resistance to enzalutamide will eventually develop. Thus, novel agents are urgently needed to treat this devastating disease. Triptolide (TPL), a key active compound extracted from the Chinese herb Thunder God Vine ( Tripterygium wilfordii Hook F.), possesses anti-cancer activity in human prostate cancer cells. However, the effects of TPL against CRPC cells and the underlying mechanism of any such effect are unknown. In this study, we found that TPL at low dose inhibits the transactivation activity of both full-length and truncated AR without changing their protein levels. Interestingly, TPL inhibits phosphorylation of AR and its CRPC-associated variant AR-V7 at Ser515 through XPB/CDK7. As a result, TPL suppresses the binding of AR to promoter regions in AR target genes along with reduced TFIIH and RNA Pol II recruitment. Moreover, TPL at low dose reduces the viability of prostate cancer cells expressing AR or AR-Vs. Low-dose TPL also shows a synergistic effect with enzalutamide to inhibit CRPC cell survival in vitro , and enhances the anti-cancer effect of enzalutamide on CRPC xenografts with minimal side effects. Taken together, our data demonstrate that TPL targets the transactivation activity of both full-length and truncated ARs. Our results also suggest that TPL is a potential drug for CRPC, and can be used in combination with enzalutamide to treat CRPC.

Triptolide Inhibits the AR Signaling Pathway to Suppress the Proliferation of Enzalutamide Resistant Prostate Cancer Cells

PubMed Central

Han, Yangyang; Huang, Weiwei; Liu, Jiakuan; Liu, Dandan; Cui, Yangyan; Huang, Ruimin; Yan, Jun; Lei, Ming

2017-01-01

Enzalutamide is a second-generation androgen receptor (AR) antagonist for the treatment of metastatic castration-resistant prostate cancer (mCRPC). Unfortunately, AR dysfunction means that resistance to enzalutamide will eventually develop. Thus, novel agents are urgently needed to treat this devastating disease. Triptolide (TPL), a key active compound extracted from the Chinese herb Thunder God Vine (Tripterygium wilfordii Hook F.), possesses anti-cancer activity in human prostate cancer cells. However, the effects of TPL against CRPC cells and the underlying mechanism of any such effect are unknown. In this study, we found that TPL at low dose inhibits the transactivation activity of both full-length and truncated AR without changing their protein levels. Interestingly, TPL inhibits phosphorylation of AR and its CRPC-associated variant AR-V7 at Ser515 through XPB/CDK7. As a result, TPL suppresses the binding of AR to promoter regions in AR target genes along with reduced TFIIH and RNA Pol II recruitment. Moreover, TPL at low dose reduces the viability of prostate cancer cells expressing AR or AR-Vs. Low-dose TPL also shows a synergistic effect with enzalutamide to inhibit CRPC cell survival in vitro, and enhances the anti-cancer effect of enzalutamide on CRPC xenografts with minimal side effects. Taken together, our data demonstrate that TPL targets the transactivation activity of both full-length and truncated ARs. Our results also suggest that TPL is a potential drug for CRPC, and can be used in combination with enzalutamide to treat CRPC. PMID:28638477

A homozygous PMS2 founder mutation with an attenuated constitutional mismatch repair deficiency phenotype.

PubMed

Li, Lili; Hamel, Nancy; Baker, Kristi; McGuffin, Michael J; Couillard, Martin; Gologan, Adrian; Marcus, Victoria A; Chodirker, Bernard; Chudley, Albert; Stefanovici, Camelia; Durandy, Anne; Hegele, Robert A; Feng, Bing-Jian; Goldgar, David E; Zhu, Jun; De Rosa, Marina; Gruber, Stephen B; Wimmer, Katharina; Young, Barbara; Chong, George; Tischkowitz, Marc D; Foulkes, William D

2015-05-01

Inherited mutations in DNA mismatch repair genes predispose to different cancer syndromes depending on whether they are mono-allelic or bi-allelic. This supports a causal relationship between expression level in the germline and phenotype variation. As a model to study this relationship, our study aimed to define the pathogenic characteristics of a recurrent homozygous coding variant in PMS2 displaying an attenuated phenotype identified by clinical genetic testing in seven Inuit families from Northern Quebec. Pathogenic characteristics of the PMS2 mutation NM_000535.5:c.2002A>G were studied using genotype-phenotype correlation, single-molecule expression detection and single genome microsatellite instability analysis. This PMS2 mutation generates a de novo splice site that competes with the authentic site. In homozygotes, expression of the full-length protein is reduced to a level barely detectable by conventional diagnostics. Median age at primary cancer diagnosis is 22 years among 13 NM_000535.5:c.2002A>G homozygotes, versus 8 years in individuals carrying bi-allelic truncating mutations. Residual expression of full-length PMS2 transcript was detected in normal tissues from homozygotes with cancers in their 20s. Our genotype-phenotype study of c.2002A>G illustrates that an extremely low level of PMS2 expression likely delays cancer onset, a feature that could be exploited in cancer preventive intervention. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

A fast ergodic algorithm for generating ensembles of equilateral random polygons

NASA Astrophysics Data System (ADS)

Varela, R.; Hinson, K.; Arsuaga, J.; Diao, Y.

2009-03-01

Knotted structures are commonly found in circular DNA and along the backbone of certain proteins. In order to properly estimate properties of these three-dimensional structures it is often necessary to generate large ensembles of simulated closed chains (i.e. polygons) of equal edge lengths (such polygons are called equilateral random polygons). However finding efficient algorithms that properly sample the space of equilateral random polygons is a difficult problem. Currently there are no proven algorithms that generate equilateral random polygons with its theoretical distribution. In this paper we propose a method that generates equilateral random polygons in a 'step-wise uniform' way. We prove that this method is ergodic in the sense that any given equilateral random polygon can be generated by this method and we show that the time needed to generate an equilateral random polygon of length n is linear in terms of n. These two properties make this algorithm a big improvement over the existing generating methods. Detailed numerical comparisons of our algorithm with other widely used algorithms are provided.

Acute effects of anesthetic lumbar spine injections on temporal spatial parameters of gait in individuals with chronic low back pain: A pilot study.

PubMed

Herndon, Carl L; Horodyski, MaryBeth; Vincent, Heather K

2017-10-01

This study examined whether epidural injection-induced anesthesia acutely and positively affected temporal spatial parameters of gait in patients with chronic low back pain (LBP) due to lumbar spinal stenosis. Twenty-five patients (61.7±13.6years) who were obtaining lumbar epidural injections for stenosis-related LBP participated. Oswestry Disability Index (ODI) scores, Medical Outcomes Short Form (SF-36) scores, 11-point Numerical pain rating (NRS pain ) scores, and temporal spatial parameters of walking gait were obtained prior to, and 11-point Numerical pain rating (NRS pain ) scores, and temporal spatial parameters of walking gait were obtained after the injection. Gait parameters were measured using an instrumented gait mat. Patients received transforaminal epidural injections in the L1-S1 vertebral range (1% lidocaine, corticosteroid) under fluoroscopic guidance. Patients with post-injection NRS pain ratings of "0" or values greater than "0" were stratified into two groups: 1) full pain relief, or 2) partial pain relief, respectively. Post-injection, 48% (N=12) of patients reported full pain relief. ODI scores were higher in patients with full pain relief (55.3±21.4 versus 33.7 12.8; p=0.008). Post-injection, stride length and step length variability were significantly improved in the patients with full pain relief compared to those with partial pain relief. Effect sizes between full and partial pain relief for walking velocity, step length, swing time, stride and step length variability were medium to large (Cohen's d>0.50). Patients with LBP can gain immediate gait improvements from complete pain relief from transforaminal epidural anesthetic injections for LBP, which could translate to better stability and lower fall risk. Copyright © 2017 Elsevier B.V. All rights reserved.

Drug resistance is conferred on the model yeast Saccharomyces cerevisiae by expression of full-length melanoma-associated human ATP-binding cassette transporter ABCB5.

PubMed

Keniya, Mikhail V; Holmes, Ann R; Niimi, Masakazu; Lamping, Erwin; Gillet, Jean-Pierre; Gottesman, Michael M; Cannon, Richard D

2014-10-06

ABCB5, an ATP-binding cassette (ABC) transporter, is highly expressed in melanoma cells, and may contribute to the extreme resistance of melanomas to chemotherapy by efflux of anti-cancer drugs. Our goal was to determine whether we could functionally express human ABCB5 in the model yeast Saccharomyces cerevisiae, in order to demonstrate an efflux function for ABCB5 in the absence of background pump activity from other human transporters. Heterologous expression would also facilitate drug discovery for this important target. DNAs encoding ABCB5 sequences were cloned into the chromosomal PDR5 locus of a S. cerevisiae strain in which seven endogenous ABC transporters have been deleted. Protein expression in the yeast cells was monitored by immunodetection using both a specific anti-ABCB5 antibody and a cross-reactive anti-ABCB1 antibody. ABCB5 function in recombinant yeast cells was measured by determining whether the cells possessed increased resistance to known pump substrates, compared to the host yeast strain, in assays of yeast growth. Three ABCB5 constructs were made in yeast. One was derived from the ABCB5-β mRNA, which is highly expressed in human tissues but is a truncation of a canonical full-size ABC transporter. Two constructs contained full-length ABCB5 sequences: either a native sequence from cDNA or a synthetic sequence codon-harmonized for S. cerevisiae. Expression of all three constructs in yeast was confirmed by immunodetection. Expression of the codon-harmonized full-length ABCB5 DNA conferred increased resistance, relative to the host yeast strain, to the putative substrates rhodamine 123, daunorubicin, tetramethylrhodamine, FK506, or clorgyline. We conclude that full-length ABCB5 can be functionally expressed in S. cerevisiae and confers drug resistance.

Direct Numerical Simulations of a Full Stationary Wind-Turbine Blade

NASA Astrophysics Data System (ADS)

Qamar, Adnan; Zhang, Wei; Gao, Wei; Samtaney, Ravi

2014-11-01

Direct numerical simulation of flow past a full stationary wind-turbine blade is carried out at Reynolds number, Re = 10,000 placed at 0 and 5 (degree) angle of attack. The study is targeted to create a DNS database for verification of solvers and turbulent models that are utilized in wind-turbine modeling applications. The full blade comprises of a circular cylinder base that is attached to a spanwise varying airfoil cross-section profile (without twist). An overlapping composite grid technique is utilized to perform these DNS computations, which permits block structure in the mapped computational space. Different flow shedding regimes are observed along the blade length. Von-Karman shedding is observed in the cylinder shaft region of the turbine blade. Along the airfoil cross-section of the blade, near body shear layer breakdown is observed. A long tip vortex originates from the blade tip region, which exits the computational plane without being perturbed. Laminar to turbulent flow transition is observed along the blade length. The turbulent fluctuations amplitude decreases along the blade length and the flow remains laminar regime in the vicinity of the blade tip. The Strouhal number is found to decrease monotonously along the blade length. Average lift and drag coefficients are also reported for the cases investigated. Supported by funding under a KAUST OCRF-CRG grant.

Narrative Analysis: Clinical Applications of Story Generation and Story Retelling.

ERIC Educational Resources Information Center

Merritt, Donna DiSegna; Liles, Betty Z.

1989-01-01

Twenty language-disordered and 20 nonimpaired children, aged 9-11, performed story generation and story retelling tasks. For both groups, retold narratives were longer and contained more story grammar components and complete episode structures. Clause length differentiated story generation from story retelling for the language-disordered children…

Non-radioactive TRF assay modifications to improve telomeric DNA detection efficiency in plants

PubMed Central

Nigmatullina, Liliia R.; Sharipova, Margarita R.; Shakirov, Eugene V.

2016-01-01

The length of telomeric DNA is often considered a cellular biomarker of aging and general health status. Several telomere length measuring assays have been developed, of which the most common is the Telomere Restriction Fragment (TRF) analysis, which typically involves the use of radioactively labeled oligonucleotide probes. While highly effective, this method potentially poses substantial health concerns and generates radioactive waste. Digoxigenin (DIG) alternatives to radioactive probes have been developed and used successfully in a number of assays. Here we optimize the DIG protocol to measure telomere length in the model plant Arabidopsis thaliana and present evidence that this approach can be used successfully to efficiently and accurately measure telomere length in plants. Specifically, hybridization temperature of 42 °C instead of the typical 55 °C appears to generate stronger signals. In addition, DIG incorporation at 5′-end instead of 3′-end of the labeled oligonucleotide greatly enhances signal. We conclude that non-radioactive TRF assays can be as efficient as radioactive methods in detecting and measuring telomere length in plants, making this assay suitable for medical and research laboratories unable to utilize radioactivity due to hazardous waste disposal and safety concerns. PMID:28133587

A new tritiated water measurement method with plastic scintillator pellets.

PubMed

Furuta, Etsuko; Iwasaki, Noriko; Kato, Yuka; Tomozoe, Yusuke

2016-01-01

A new tritiated water measurement method with plastic scintillator pellets (PS-pellets) by using a conventional liquid scintillation counter was developed. The PS-pellets used were 3 mm in both diameter and length. A low potassium glass vial was filled full with the pellets, and tritiated water was applied to the vial from 5 to 100 μl. Then, the sample solution was scattered in the interstices of the pellets in a vial. This method needs no liquid scintillator, so no liquid organic waste fluid is generated. The counting efficiency with the pellets was approximately 48 % when a 5 μl solution was used, which was higher than that of conventional measurement using liquid scintillator. The relationship between count rate and activity showed good linearity. The pellets were able to be used repeatedly, so few solid wastes are generated with this method. The PS-pellets are useful for tritiated water measurement; however, it is necessary to develop a new device which can be applied to a larger volume and measure low level concentration like an environmental application.

Cloning and construction of recombinant palI gene from Klebsiella oxytoca on pET-32b into E. coli BL21 (DE3) pLysS for production of isomaltulose, a new generation of sugar

DOE Office of Scientific and Technical Information (OSTI.GOV)

Moeis, Maelita R., E-mail: sony@sith.itb.ac.id; Berlian, Liska, E-mail: sony@sith.itb.ac.id; Suhandono, Sony, E-mail: sony@sith.itb.ac.id

Klebsiella oxytoca produces sucrose isomerase which catalyses the conversion of sucrose to isomaltulose, a new generation of sugar. From the previous study, palI gene from Klebsiella oxytoca was succesfully isolated from sapodilla fruit (Manilkara zapota). The full-length palI gene sequence of Klebsiella oxytoca was cloned in E. coli DH5α. The deduced amino acid sequence shows 498 residues which includes conserved motif for sucrose isomerisation {sup 325}RLDRD{sup 329} and 97% identical to palI gene from Klebsiella sp. LX3 (GenBank:AAK82938.1). This fragment was succesfullly ligated into the expression vector pET-32b using overlap-extension PCR and cloned in Escherichia coli BL21 (DE3) pLysS. DNAmore » sequencing result shows that palI gene of Klebsiella oxytoca was inserted in-frame in pET-32b. This is the first report on cloning of palI gene from Klebsiella oxytoca.« less

Functional Analysis With a Barcoder Yeast Gene Overexpression System

PubMed Central

Douglas, Alison C.; Smith, Andrew M.; Sharifpoor, Sara; Yan, Zhun; Durbic, Tanja; Heisler, Lawrence E.; Lee, Anna Y.; Ryan, Owen; Göttert, Hendrikje; Surendra, Anu; van Dyk, Dewald; Giaever, Guri; Boone, Charles; Nislow, Corey; Andrews, Brenda J.

2012-01-01

Systematic analysis of gene overexpression phenotypes provides an insight into gene function, enzyme targets, and biological pathways. Here, we describe a novel functional genomics platform that enables a highly parallel and systematic assessment of overexpression phenotypes in pooled cultures. First, we constructed a genome-level collection of ~5100 yeast barcoder strains, each of which carries a unique barcode, enabling pooled fitness assays with a barcode microarray or sequencing readout. Second, we constructed a yeast open reading frame (ORF) galactose-induced overexpression array by generating a genome-wide set of yeast transformants, each of which carries an individual plasmid-born and sequence-verified ORF derived from the Saccharomyces cerevisiae full-length EXpression-ready (FLEX) collection. We combined these collections genetically using synthetic genetic array methodology, generating ~5100 strains, each of which is barcoded and overexpresses a specific ORF, a set we termed “barFLEX.” Additional synthetic genetic array allows the barFLEX collection to be moved into different genetic backgrounds. As a proof-of-principle, we describe the properties of the barFLEX overexpression collection and its application in synthetic dosage lethality studies under different environmental conditions. PMID:23050238

Quetzal: a transposon of the Tc1 family in the mosquito Anopheles albimanus.

PubMed

Ke, Z; Grossman, G L; Cornel, A J; Collins, F H

1996-10-01

A member of the Tc1 family of transposable elements has been identified in the Central and South American mosquito Anopheles albimanus. The full-length Quetzal element is 1680 base pairs (bp) in length, possesses 236 bp inverted terminal repeats (ITRs), and has a single open reading frame (ORF) with the potential of encoding a 341-amino-acid (aa) protein that is similar to the transposases of other members of the Tc1 family, particularly elements described from three different Drosophila species. The approximately 10-12 copies per genome of Quetzal are found in the euchromatin of all three chromosomes of A. albimanus. One full-length clone, Que27, appears capable of encoding a complete transposase and may represent a functional copy of this element.

Development of Transcriptomic Resources for Interrogating the Biosynthesis of Monoterpene Indole Alkaloids in Medicinal Plant Species

PubMed Central

Góngora-Castillo, Elsa; Childs, Kevin L.; Fedewa, Greg; Hamilton, John P.; Liscombe, David K.; Magallanes-Lundback, Maria; Mandadi, Kranthi K.; Nims, Ezekiel; Runguphan, Weerawat; Vaillancourt, Brieanne; Varbanova-Herde, Marina; DellaPenna, Dean; McKnight, Thomas D.; O’Connor, Sarah; Buell, C. Robin

2012-01-01

The natural diversity of plant metabolism has long been a source for human medicines. One group of plant-derived compounds, the monoterpene indole alkaloids (MIAs), includes well-documented therapeutic agents used in the treatment of cancer (vinblastine, vincristine, camptothecin), hypertension (reserpine, ajmalicine), malaria (quinine), and as analgesics (7-hydroxymitragynine). Our understanding of the biochemical pathways that synthesize these commercially relevant compounds is incomplete due in part to a lack of molecular, genetic, and genomic resources for the identification of the genes involved in these specialized metabolic pathways. To address these limitations, we generated large-scale transcriptome sequence and expression profiles for three species of Asterids that produce medicinally important MIAs: Camptotheca acuminata, Catharanthus roseus, and Rauvolfia serpentina. Using next generation sequencing technology, we sampled the transcriptomes of these species across a diverse set of developmental tissues, and in the case of C. roseus, in cultured cells and roots following elicitor treatment. Through an iterative assembly process, we generated robust transcriptome assemblies for all three species with a substantial number of the assembled transcripts being full or near-full length. The majority of transcripts had a related sequence in either UniRef100, the Arabidopsis thaliana predicted proteome, or the Pfam protein domain database; however, we also identified transcripts that lacked similarity with entries in either database and thereby lack a known function. Representation of known genes within the MIA biosynthetic pathway was robust. As a diverse set of tissues and treatments were surveyed, expression abundances of transcripts in the three species could be estimated to reveal transcripts associated with development and response to elicitor treatment. Together, these transcriptomes and expression abundance matrices provide a rich resource for understanding plant specialized metabolism, and promotes realization of innovative production systems for plant-derived pharmaceuticals. PMID:23300689

A near full-length open reading frame next generation sequencing assay for genotyping and identification of resistance-associated variants in hepatitis C virus.

PubMed

Pedersen, M S; Fahnøe, U; Hansen, T A; Pedersen, A G; Jenssen, H; Bukh, J; Schønning, K

2018-06-01

The current treatment options for hepatitis C virus (HCV), based on direct acting antivirals (DAA), are dependent on virus genotype and previous treatment experience. Treatment failures have been associated with detection of resistance-associated substitutions (RASs) in the DAA targets of HCV, the NS3, NS5A and NS5 B proteins. To develop a next generation sequencing based method that provides genotype and detection of HCV NS3, NS5A, and NS5 B RASs without prior knowledge of sample genotype. In total, 101 residual plasma samples from patients with HCV covering 10 different viral subtypes across 4 genotypes with viral loads of 3.84-7.61 Log IU/mL were included. All samples were de-identified and consequently prior treatment status for patients was unknown. Almost full open reading frame amplicons (∼ 9 kb) were generated using RT-PCR with a single primer set. The resulting amplicons were sequenced with high throughput sequencing and analysed using an in-house developed script for detecting RASs. The method successfully amplified and sequenced 94% (95/101) of samples with an average coverage of 14,035; four of six failed samples were genotype 4a. Samples analysed twice yielded reproducible nucleotide frequencies across all sites. RASs were detected in 21/95 (22%) samples at a 15% threshold. The method identified one patient infected with two genotype 2b variants, and the presence of subgenomic deletion variants in 8 (8.4%) of 95 successfully sequenced samples. The presented method may provide identification of HCV genotype, RASs detection, and detect multiple HCV infection without prior knowledge of sample genotype. Copyright © 2018 Elsevier B.V. All rights reserved.

Peptides having reduced toxicity that stimulate cholesterol efflux

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bielicki, John K.; Johansson, Jan; Danho, Waleed

The present invention provides a family of non-naturally occurring polypeptides having cholesterol efflux activity that parallels that of full-length apolipoproteins (e.g., Apo AI and Apo E), and having high selectivity for ABCA1 that parallels that of full-length apolipoproteins. Further, the peptides of the invention have little or no toxicity when administered at therapeutic and higher doses. The invention also provides compositions comprising such polypeptides, methods of identifying, screening and synthesizing such polypeptides, and methods of treating, preventing or diagnosing diseases and disorders associated with dyslipidemia, hypercholesterolemia and inflammation.

Optimization of mid-IR generation from a periodically poled MgO doped stoichiometric lithium tantalate optical parametric oscillator with intracavity difference frequency mixing

NASA Astrophysics Data System (ADS)

Hatano, Hideki; Slater, Richard; Takekawa, Shunji; Kusano, Masahiro; Watanabe, Makoto

2017-07-01

We demonstrate 43% slope efficiency for generation of ∼3200 nm light, a wavelength considered to be ideal for laser induced ultrasound generation in carbon fiber reinforced plastic. High slope efficiency was obtained by optimizing crystal lengths, cavity length and mirror reflectivity using a two crystal optical parametric oscillator+difference frequency mixing (OPO+DFM) nonlinear wavelength conversion scheme. Mid-IR output >12 mJ was obtained from a 1064 nm Nd:YAG pump laser with 12 ns pulse width (FWHM) and containing pulse energy of 43 mJ. A compact, single temperature crystal oven is described along with some suggestions for improving the slope efficiency.

The impact of severe obesity on hospital length of stay.

PubMed

Hauck, Katharina; Hollingsworth, Bruce

2010-04-01

The excess health care costs caused by obesity are a concern in many countries, yet little is known about the additional resources required to treat obese patients in hospitals. To estimate differences in hospital resource use, measured by length of stay, between severely obese and other patients, conditioning on a range of patient and hospital characteristics. Administrative patient-level hospital data for 122 Australian public hospitals over the financial year 2005/06 (Victorian Admitted Episodes Data). Episodes (435,147) for patients above 17 years of age and with a stay of one night or more. Quantile Regression analysis is used to generate 19 estimates of the difference between severely obese and other patients across the whole range of length of stay, from very short to very long staying patients. Separate estimates for 17 hospital specialties and for medically and surgically treated patients are generated. There are significant differences in average length of stay for almost all specialties. For some, differences are less than 1 day, but for others, severely obese patients stay up to 4 days longer. For a number of specialties, obese patients have significantly shorter length of stay. Overall, medically managed obese patients stay longer, whereas surgically treated patients stay shorter than other patients. Differences tend to increase with length of stay. Differences in length of stay may arise because severely obese patients are medically more complex. The observed shorter stays for obese patients in some specialties may result from their observed greater likelihood of being transferred to another hospital.

A Universal Next-Generation Sequencing Protocol To Generate Noninfectious Barcoded cDNA Libraries from High-Containment RNA Viruses

PubMed Central

Moser, Lindsey A.; Ramirez-Carvajal, Lisbeth; Puri, Vinita; Pauszek, Steven J.; Matthews, Krystal; Dilley, Kari A.; Mullan, Clancy; McGraw, Jennifer; Khayat, Michael; Beeri, Karen; Yee, Anthony; Dugan, Vivien; Heise, Mark T.; Frieman, Matthew B.; Rodriguez, Luis L.; Bernard, Kristen A.; Wentworth, David E.

2016-01-01

ABSTRACT Several biosafety level 3 and/or 4 (BSL-3/4) pathogens are high-consequence, single-stranded RNA viruses, and their genomes, when introduced into permissive cells, are infectious. Moreover, many of these viruses are select agents (SAs), and their genomes are also considered SAs. For this reason, cDNAs and/or their derivatives must be tested to ensure the absence of infectious virus and/or viral RNA before transfer out of the BSL-3/4 and/or SA laboratory. This tremendously limits the capacity to conduct viral genomic research, particularly the application of next-generation sequencing (NGS). Here, we present a sequence-independent method to rapidly amplify viral genomic RNA while simultaneously abolishing both viral and genomic RNA infectivity across multiple single-stranded positive-sense RNA (ssRNA+) virus families. The process generates barcoded DNA amplicons that range in length from 300 to 1,000 bp, which cannot be used to rescue a virus and are stable to transport at room temperature. Our barcoding approach allows for up to 288 barcoded samples to be pooled into a single library and run across various NGS platforms without potential reconstitution of the viral genome. Our data demonstrate that this approach provides full-length genomic sequence information not only from high-titer virion preparations but it can also recover specific viral sequence from samples with limited starting material in the background of cellular RNA, and it can be used to identify pathogens from unknown samples. In summary, we describe a rapid, universal standard operating procedure that generates high-quality NGS libraries free of infectious virus and infectious viral RNA. IMPORTANCE This report establishes and validates a standard operating procedure (SOP) for select agents (SAs) and other biosafety level 3 and/or 4 (BSL-3/4) RNA viruses to rapidly generate noninfectious, barcoded cDNA amenable for next-generation sequencing (NGS). This eliminates the burden of testing all processed samples derived from high-consequence pathogens prior to transfer from high-containment laboratories to lower-containment facilities for sequencing. Our established protocol can be scaled up for high-throughput sequencing of hundreds of samples simultaneously, which can dramatically reduce the cost and effort required for NGS library construction. NGS data from this SOP can provide complete genome coverage from viral stocks and can also detect virus-specific reads from limited starting material. Our data suggest that the procedure can be implemented and easily validated by institutional biosafety committees across research laboratories. PMID:27822536

Characterization of L1 ORF1p self-interaction and cellular localization using a mammalian two-hybrid system.

PubMed

Sokolowski, Mark; deHaro, Dawn; Christian, Claiborne M; Kines, Kristine J; Belancio, Victoria P

2013-01-01

Long INterspersed Element-1 (LINE-1, L1) is an active retrotransposon that mobilizes using a ribonucleoprotein particle (RNP) intermediate composed of the full-length bicistronic L1 mRNA and the two proteins (ORF1p and ORF2p) encoded by that mRNA. ORF1p and ORF2p demonstrate cis-preference for their encoding mRNA. Previous studies of ORF1p, purified from bacterial and insect cells demonstrated that this protein forms trimers in vitro. While valuable for understanding ORF1p function, these in vitro approaches do not provide any information on ORF1p self-interaction in the context of mammalian cells. We used a mammalian two-hybrid (M2H) system in order to study L1 ORF1p self-interaction in human and mouse cells. We demonstrate that the M2H system successfully detects human and mouse ORF1p self-interactions in transiently transfected mammalian cells. We also generated mouse and human ORF1p-specific antibodies to characterize the expression of ORF1p fusion proteins used in the M2H system. Using these antibodies, we demonstrate that ORF1p interaction in trans leads to the formation of heterodimers that are expected to produce a positive signal in the M2H system. Although the role for L1 ORF1p cis-preference in L1 mobilization is established, the impact of ability of ORF1pto interact in trans on the L1 replication cycle is not known. Furthermore, western blot analysis of ORF1p generated by a full-length L1, wild type ORF1, or a codon-optimized ORF1 expression vector is detected in the nucleus. In contrast, the addition of a tag to the N-terminus of the mouse and human ORF1 proteins can significantly alter the subcellular localization in a tag-specific manner. These data support that nuclear localization of ORF1p may contribute to L1 (and potentially the SINE Alu) RNP nuclear access in the host cell.

Construction of C35 gene bait recombinants and T47D cell cDNA library.

PubMed

Yin, Kun; Xu, Chao; Zhao, Gui-Hua; Liu, Ye; Xiao, Ting; Zhu, Song; Yan, Ge

2017-11-20

C35 is a novel tumor biomarker associated with metastasis progression. To investigate the interaction factors of C35 in its high expressed breast cancer cell lines, we constructed bait recombinant plasmids of C35 gene and T47D cell cDNA library for yeast two-hybrid screening. Full length C35 sequences were subcloned using RT-PCR from cDNA template extracted from T47D cells. Based on functional domain analysis, the full-length C35 1-348bp was also truncated into two fragments C351-153bp and C35154-348bp to avoid auto-activation. The three kinds of C35 genes were successfully amplified and inserted into pGBKT7 to construct bait recombinant plasmids pGBKT7-C351-348bp, pGBKT7-C351-153bp and pGBKT7-C35154-348bp, then transformed into Y187 yeast cells by the lithium acetate method. Auto-activation and toxicity of C35 baits were detected using nutritional deficient medium and X-α-Gal assays. The T47D cell ds cDNA was generated by SMART TM technology and the library was constructed using in vivo recombination-mediated cloning in the AH109 yeast strain using a pGADT7-Rec plasmid. The transformed Y187/pGBKT7-C351-348bp line was intensively inhibited while the truncated Y187/pGBKT7-C35 lines had no auto-activation and toxicity in yeast cells. The titer of established cDNA library was 2 × 10 7 pfu/mL with high transformation efficiency of 1.4 × 10 6 , and the insert size of ds cDNA was distributed homogeneously between 0.5-2.0 kb. Our research generated a T47D cell cDNA library with high titer, and the constructed two C35 "baits" contained a respective functional immunoreceptor tyrosine based activation motif (ITAM) and the conserved last four amino acids Cys-Ile-Leu-Val (CILV) motif, and therefore laid a foundation for screening the C35 interaction factors in a BC cell line.

Evaluation of a genetically modified foot-and-mouth disease virus vaccine candidate generated by reverse genetics

PubMed Central

2012-01-01

Background Foot-and-mouth disease (FMD) is the most economically important and highly contagious disease of cloven-hoofed animals worldwide. Control of the disease has been mainly based on large-scale vaccinations with whole-virus inactivated vaccines. In recent years, a series of outbreaks of type O FMD occurred in China (including Chinese Taipei, Chinese Hong Kong) posed a tremendous threat to Chinese animal husbandry. Its causative agent, type O FMDV, has evolved into three topotypes (East–South Asia (ME-SA), Southeast Asia (SEA), Cathay (CHY)) in these regions, which represents an important obstacle to disease control. The available FMD vaccine in China shows generally good protection against ME-SA and SEA topotype viruses infection, but affords insufficient protection against some variants of the CHY topotype. Therefore, the choice of a new vaccine strain is of fundamental importance. Results The present study describes the generation of a full-length infectious cDNA clone of FMDV vaccine strain and a genetically modified virus with some amino acid substitutions in antigenic sites 1, 3, and 4, based on the established infectious clone. The recombinant viruses had similar growth properties to the wild O/HN/CHA/93 virus. All swine immunized with inactivated vaccine prepared from the O/HN/CHA/93 were fully protected from challenge with the viruses of ME-SA and SEA topotypes and partially protected against challenge with the virus of CHY topotype at 28 days post-immunization. In contrast, the swine inoculated with the genetically modified vaccine were completely protected from the infection of viruses of the three topotypes. Conclusions Some amino acid substitutions in the FMDV vaccine strain genome did not have an effect on the ability of viral replication in vitro. The vaccine prepared from genetically modified FMDV by reverse genetics significantly improved the protective efficacy to the variant of the CHY topotype, compared with the wild O/HN/CHA/93 virus. Thus, the full-length cDNA clone of FMDV can be a useful tool to develop genetically engineered FMDV vaccine candidates to help control porcinophilic FMD epidemics in China. PMID:22591597

Analysis and elimination of a bias in targeted molecular dynamics simulations of conformational transitions: application to calmodulin.

PubMed

Ovchinnikov, Victor; Karplus, Martin

2012-07-26

The popular targeted molecular dynamics (TMD) method for generating transition paths in complex biomolecular systems is revisited. In a typical TMD transition path, the large-scale changes occur early and the small-scale changes tend to occur later. As a result, the order of events in the computed paths depends on the direction in which the simulations are performed. To identify the origin of this bias, and to propose a method in which the bias is absent, variants of TMD in the restraint formulation are introduced and applied to the complex open ↔ closed transition in the protein calmodulin. Due to the global best-fit rotation that is typically part of the TMD method, the simulated system is guided implicitly along the lowest-frequency normal modes, until the large spatial scales associated with these modes are near the target conformation. The remaining portion of the transition is described progressively by higher-frequency modes, which correspond to smaller-scale rearrangements. A straightforward modification of TMD that avoids the global best-fit rotation is the locally restrained TMD (LRTMD) method, in which the biasing potential is constructed from a number of TMD potentials, each acting on a small connected portion of the protein sequence. With a uniform distribution of these elements, transition paths that lack the length-scale bias are obtained. Trajectories generated by steered MD in dihedral angle space (DSMD), a method that avoids best-fit rotations altogether, also lack the length-scale bias. To examine the importance of the paths generated by TMD, LRTMD, and DSMD in the actual transition, we use the finite-temperature string method to compute the free energy profile associated with a transition tube around a path generated by each algorithm. The free energy barriers associated with the paths are comparable, suggesting that transitions can occur along each route with similar probabilities. This result indicates that a broad ensemble of paths needs to be calculated to obtain a full description of conformational changes in biomolecules. The breadth of the contributing ensemble suggests that energetic barriers for conformational transitions in proteins are offset by entropic contributions that arise from a large number of possible paths.

Horizontal visibility graphs generated by type-I intermittency

NASA Astrophysics Data System (ADS)

Núñez, Ángel M.; Luque, Bartolo; Lacasa, Lucas; Gómez, Jose Patricio; Robledo, Alberto

2013-05-01

The type-I intermittency route to (or out of) chaos is investigated within the horizontal visibility (HV) graph theory. For that purpose, we address the trajectories generated by unimodal maps close to an inverse tangent bifurcation and construct their associated HV graphs. We show how the alternation of laminar episodes and chaotic bursts imprints a fingerprint in the resulting graph structure. Accordingly, we derive a phenomenological theory that predicts quantitative values for several network parameters. In particular, we predict that the characteristic power-law scaling of the mean length of laminar trend sizes is fully inherited by the variance of the graph degree distribution, in good agreement with the numerics. We also report numerical evidence on how the characteristic power-law scaling of the Lyapunov exponent as a function of the distance to the tangent bifurcation is inherited in the graph by an analogous scaling of block entropy functionals defined on the graph. Furthermore, we are able to recast the full set of HV graphs generated by intermittent dynamics into a renormalization-group framework, where the fixed points of its graph-theoretical renormalization-group flow account for the different types of dynamics. We also establish that the nontrivial fixed point of this flow coincides with the tangency condition and that the corresponding invariant graph exhibits extremal entropic properties.

Adenoviral vectors elicit humoral immunity against variable loop 2 of clade C HIV-1 gp120 via "Antigen Capsid-Incorporation" strategy.

PubMed

Gu, Linlin; Krendelchtchikova, Valentina; Krendelchtchikov, Alexandre; Farrow, Anitra L; Derdeyn, Cynthia A; Matthews, Qiana L

2016-01-01

Adenoviral (Ad) vectors in combination with the "Antigen Capsid-Incorporation" strategy have been applied in developing HIV-1 vaccines, due to the vectors׳ abilities in incorporating and inducing immunity of capsid-incorporated antigens. Variable loop 2 (V2)-specific antibodies were suggested in the RV144 trial to correlate with reduced HIV-1 acquisition, which highlights the importance of developing novel HIV-1 vaccines by targeting the V2 loop. Therefore, the V2 loop of HIV-1 has been incorporated into the Ad capsid protein. We generated adenovirus serotype 5 (Ad5) vectors displaying variable loop 2 (V2) of HIV-1 gp120, with the "Antigen Capsid-Incorporation" strategy. To assess the incorporation capabilities on hexon hypervariable region1 (HVR1) and protein IX (pIX), 20aa or full length (43aa) of V2 and V1V2 (67aa) were incorporated, respectively. Immunizations with the recombinant vectors significantly generated antibodies against both linear and discontinuous V2 epitopes. The immunizations generated durable humoral immunity against V2. This study will lead to more stringent development of various serotypes of adenovirus-vectored V2 vaccine candidates, based on breakthroughs regarding the immunogenicity of V2. Copyright © 2015. Published by Elsevier Inc.

Design study of piezoelectric energy-harvesting devices for generation of higher electrical power using a coupled piezoelectric-circuit finite element method.

PubMed

Zhu, Meiling; Worthington, Emma; Tiwari, Ashutosh

2010-01-01

This paper presents a design study on the geometric parameters of a cantilever-based piezoelectric energy-harvesting devices (EHD), which harvest energy from motion (vibration), for the purpose of scavenging more energy from ambient vibration energy sources. The design study is based on the coupled piezoelectric-circuit finite element method (CPCFEM), previously presented by Dr. Zhu. This model can calculate the power output of piezoelectric EHDS directly connected to a load resistor and is used in this paper to obtain the following simulation results for variations in geometric parameters such as the beam length, width and thickness, and the mass length, width, and height: 1) the current flowing through and the voltage developed across the load resistor, 2) the power dissipated by the resistor and the corresponding vibrational displacement amplitude, and 3) the resonant frequency. By studying these results, straightforward design strategies that enable the generation of more power are obtained for each geometric parameter, and a physical understanding of how each parameter affects the output power is given. It is suggested that, in designing with the aim of generating more power, the following strategies be used: 1) for the beam, a shorter length, larger width, and lower ratio of piezoelectric layer thickness to total beam thickness are preferred in the case of a fixed mass; 2) for the mass, a shortened mass length and a higher mass height are preferred in the case of variation in the mass length and the mass height with mass width and mass value remain fixed, and a wider width and small mass height are preferred in the case of variation in mass width and height (mass length and value remain fixed; and 3) for the case of a fixed total length, a shorter beam length and longer mass length are preferred. With the design strategies, output powers from the device can reach above 1 to 2 mW/cm(3), much higher than the 200 microW/cm(3) currently achieved in the published literature. This is an encouraging prospect for enabling a wider range of applications of the EHDs. In addition, physical insights into how each parameter influences output power are also discussed in detail.

Generation and Characterization of a Defective HIV-1 Virus as an Immunogen for a Therapeutic Vaccine

PubMed Central

García-Pérez, Javier; García, Felipe; Blanco, Julia; Escribà-García, Laura; Gatell, Jose Maria; Alcamí, Jose; Plana, Montserrat; Sánchez-Palomino, Sonsoles

2012-01-01

Background The generation of new immunogens able to elicit strong specific immune responses remains a major challenge in the attempts to obtain a prophylactic or therapeutic vaccine against HIV/AIDS. We designed and constructed a defective recombinant virus based on the HIV-1 genome generating infective but non-replicative virions able to elicit broad and strong cellular immune responses in HIV-1 seropositive individuals. Results Viral particles were generated through transient transfection in producer cells (293-T) of a full length HIV-1 DNA carrying a deletion of 892 base pairs (bp) in the pol gene encompassing the sequence that codes for the reverse transcriptase (NL4-3/ΔRT clone). The viral particles generated were able to enter target cells, but due to the absence of reverse transcriptase no replication was detected. The immunogenic capacity of these particles was assessed by ELISPOT to determine γ-interferon production in a cohort of 69 chronic asymptomatic HIV-1 seropositive individuals. Surprisingly, defective particles produced from NL4-3/ΔRT triggered stronger cellular responses than wild-type HIV-1 viruses inactivated with Aldrithiol-2 (AT-2) and in a larger proportion of individuals (55% versus 23% seropositive individuals tested). Electron microscopy showed that NL4-3/ΔRT virions display immature morphology. Interestingly, wild-type viruses treated with Amprenavir (APV) to induce defective core maturation also induced stronger responses than the same viral particles generated in the absence of protease inhibitors. Conclusions We propose that immature HIV-1 virions generated from NL4-3/ΔRT viral clones may represent new prototypes of immunogens with a safer profile and stronger capacity to induce cellular immune responses than wild-type inactivated viral particles. PMID:23144996

Sequence Data for Clostridium autoethanogenum using Three Generations of Sequencing Technologies

DOE PAGES

Utturkar, Sagar M.; Klingeman, Dawn Marie; Bruno-Barcena, José M.; ...

2015-04-14

During the past decade, DNA sequencing output has been mostly dominated by the second generation sequencing platforms which are characterized by low cost, high throughput and shorter read lengths for example, Illumina. The emergence and development of so called third generation sequencing platforms such as PacBio has permitted exceptionally long reads (over 20 kb) to be generated. Due to read length increases, algorithm improvements and hybrid assembly approaches, the concept of one chromosome, one contig and automated finishing of microbial genomes is now a realistic and achievable task for many microbial laboratories. In this paper, we describe high quality sequencemore » datasets which span three generations of sequencing technologies, containing six types of data from four NGS platforms and originating from a single microorganism, Clostridium autoethanogenum. The dataset reported here will be useful for the scientific community to evaluate upcoming NGS platforms, enabling comparison of existing and novel bioinformatics approaches and will encourage interest in the development of innovative experimental and computational methods for NGS data.« less

An evolution based biosensor receptor DNA sequence generation algorithm.

PubMed

Kim, Eungyeong; Lee, Malrey; Gatton, Thomas M; Lee, Jaewan; Zang, Yupeng

2010-01-01

A biosensor is composed of a bioreceptor, an associated recognition molecule, and a signal transducer that can selectively detect target substances for analysis. DNA based biosensors utilize receptor molecules that allow hybridization with the target analyte. However, most DNA biosensor research uses oligonucleotides as the target analytes and does not address the potential problems of real samples. The identification of recognition molecules suitable for real target analyte samples is an important step towards further development of DNA biosensors. This study examines the characteristics of DNA used as bioreceptors and proposes a hybrid evolution-based DNA sequence generating algorithm, based on DNA computing, to identify suitable DNA bioreceptor recognition molecules for stable hybridization with real target substances. The Traveling Salesman Problem (TSP) approach is applied in the proposed algorithm to evaluate the safety and fitness of the generated DNA sequences. This approach improves efficiency and stability for enhanced and variable-length DNA sequence generation and allows extension to generation of variable-length DNA sequences with diverse receptor recognition requirements.

An in vitro evaluation of the influence of neonatal endotracheal tube diameter and length on the work of breathing.

PubMed

Miyake, Fuyu; Suga, Rika; Akiyama, Takahiro; Namba, Fumihiko

2018-04-06

Neonates, particularly premature babies, are often managed with endotracheal intubation and subsequent mechanical ventilation to maintain adequate pulmonary gas exchange. There is no consensus on the standard length of endotracheal tube. Although a short tube reduces resistance and respiratory dead space, it is believed to increase the risk of accidental extubation. There are not entirely coherent data regarding the effect of endotracheal tube length on work of breathing in infants. The aim of this study was to evaluate the impact of neonatal endotracheal tube diameter and length on the work of breathing using an infant in vitro lung model. We assessed the work of breathing index and mechanical ventilation settings with various endotracheal tube diameters and lengths using the JTR100 in vitro infant lung model. The basic parameters of the model were breathing frequency of 20 per minutes, inspiratory-expiratory ratio of 1:3, and positive end-expiratory pressure of 5 cmH 2 O. In addition, the diaphragm driving pressure to maintain the set tidal volume was measured as the work of breathing index. The JTR100 was connected to the Babylog 8000plus through the endotracheal tube. Finally, we monitored the peak inspiratory pressure generated during assist-control volume guarantee mode with a targeted tidal volume of 10-30 mL. The diaphragm driving pressure using a 2.0-mm inner diameter tube was twice as high as that using a 4.0-mm inner diameter tube. To maintain the targeted tidal volume, a shorter tube reduced both the diaphragm driving pressure and ventilator-generated peak inspiratory pressure. The difference in the generated peak inspiratory pressure between the shortest and longest tubes was 5 cmH 2 O. In our infant lung model, a shorter tube resulted in a lower work of breathing and lower ventilator-generated peak inspiratory pressure. © 2018 John Wiley & Sons Ltd.

Investigation of laser pulse length and pre-plasma scale length impact on hot electron generation on OMEGA-EP

DOE Office of Scientific and Technical Information (OSTI.GOV)

Peebles, J.; Wei, M. S.; Arefiev, A. V.

A series of experiments studying pre-plasma’s effect on electron generation and transport due to a high intensity laser were conducted on the OMEGA-EP laser facility. A controlled pre-plasma was produced in front of an aluminum foil target prior to the arrival of the high intensity short pulse beam. Energetic electron spectra were characterized with magnetic and bremsstrahlung spectrometers. Preplasma and pulse length were shown to have a large impact on the temperature of lower energy, ponderomotive scaling electrons. Furthermore, super-ponderomotive electrons, seen in prior pre-plasma experiments with shorter pulses, were observed without any initial pre-plasma in our experiment. 2D particle-in-cellmore » and radiation-hydrodynamic simulations shed light on and validate these experimental results.« less

Investigation of laser pulse length and pre-plasma scale length impact on hot electron generation on OMEGA-EP

DOE PAGES

Peebles, J.; Wei, M. S.; Arefiev, A. V.; ...

2017-02-02

A series of experiments studying pre-plasma’s effect on electron generation and transport due to a high intensity laser were conducted on the OMEGA-EP laser facility. A controlled pre-plasma was produced in front of an aluminum foil target prior to the arrival of the high intensity short pulse beam. Energetic electron spectra were characterized with magnetic and bremsstrahlung spectrometers. Preplasma and pulse length were shown to have a large impact on the temperature of lower energy, ponderomotive scaling electrons. Furthermore, super-ponderomotive electrons, seen in prior pre-plasma experiments with shorter pulses, were observed without any initial pre-plasma in our experiment. 2D particle-in-cellmore » and radiation-hydrodynamic simulations shed light on and validate these experimental results.« less